Biophotonic compositions, methods, and kits for inhibiting and disrupting biofilms

ABSTRACT

The present document describes methods of using compositions for inhibiting biofilm formation, or disrupting existing or developing biofilms in a subject, which composition comprises at least one chromophore and a pharmacologically acceptable carrier.

TECHNICAL FIELD

This description relates to the field of inhibiting biofilm formation ordisrupting existing or developing biofilms in a subject. Said biofilmscould be associated with skin or soft tissues in a medical condition.

BACKGROUND

Biofilms are mucilaginous communities of microorganisms such asbacteria, archaea, fungi, molds, algae or protozoa or mixtures thereofthat grow on various surfaces. Biofilms form when microorganismsestablish themselves on a surface and activate genes involved inproducing a matrix that includes polysaccharides. This matrix mayprovide protection of biofilm forming bacteria from biocides.

Biofilms can be formed by different microorganism species and generaincluding bacteria, archaea, fungi, molds, algae or protozoa or mixturesthereof that grow on various surfaces. Current therapies to treattissue-related biofilms include debridement, systemic antibiotics,topical agents, and bacteriophages. Debridement of wounds is performedto mechanically remove and disperse the biofilm. Based on the severityof wounds, simple tools such as curette or ultrasound energy are used todisrupt the biofilm matrix. While this technique may disrupt the ECM ofthe biofilm, it will not prevent further growth of bacteria in thewound. Thus, treatment of tissue-related biofilms often combinesdebridement of wounds with other antimicrobial treatments.

Systemic antibiotics are used as a systemic, systems-wide treatment ofthe biofilm and may prevent further growth of bacteria on the woundsurface. While systemic antibiotics may retard biofilm growth to anextent, elimination of the microorganisms forming the biofilm wouldrequire antibiotics at 500-1000 times the level that can be achievedusing systemic antibiotics. Thus, topical antibiotic treatment has beenthe standard of care for biofilms.

Topical agents such as antibiotic creams, silver, cadexomer iodine, andmethylene blue are topically applied to treat the biofilm. The advantageof using topical antibiotics to treat biofilms is that the concentrationused on the biofilm is much higher than that which can be achieved usingsystemic antibiotics. However, the inability of topical antibiotics topenetrate through the biofilm matrix is a key factor in the antibioticresistance commonly found in biofilms.

While strong antimicrobial agents may kill bacteria at the surface ofthe biofilm, bacteria at the center of the biofilm, which are protectedby the matrix, might be illuminated with a low or non-lethalconcentration of antimicrobial agents. It has been well documented thatthe penetration rates of antibiotics through biofilms are dependent notonly on the bacterial species forming the biofilm, but also on theantibiotic used. Agents that help antimicrobials penetrate the biofilm'ssurface layer will improve the effectiveness of the antimicrobials.Another factor explaining the high resistance rate of biofilms toantibiotics is due to the dense, low-oxygen environment created bybiofilms. The low-oxygen levels result in decreased metabolic activity,and stagnated growth rates of bacteria. Since many antibiotics such asβ-lactam antibiotics are only toxic against actively growing bacteria,the decreased growth rate contributes to antibiotic resistance.

There is, therefore, an ongoing need to identify agents, compositionsand methods for inhibiting biofilm formation or disrupting existing ordeveloping biofilms. The biophotonic therapy of the disclosure providesnew methods for inhibiting biofilm formation or disrupting existing ordeveloping biofilms, such as biofilms related to skin or soft tissues orbiofilms associated with a medical condition in a subject, such aswounds.

SUMMARY OF THE INVENTION

The present disclosure describes methods, compositions, kits, and usesfor inhibiting biofilm formation, disrupting existing biofilms,disrupting developing biofilms, and/or breaking down biofilms, such asthose found in wounds, skin, or soft tissues of a subject. In someembodiments, the biofilm comprises Staphylococcus aureus and/orPseudomonas aeruginosa. In some embodiments, the biofilm is produced bya microorganism such as bacteria, yeast, or fungi. A subject in need canapply any composition as described herein directly to the area ofbiofilm.

In one aspect, this disclosure describes a method for inhibiting biofilmformation or disrupting existing or developing biofilms in a subject,comprising: a) topically applying a composition comprising at least onechromophore and a pharmaceutically acceptable carrier; and b)illuminating said composition with actinic light, thereby inhibitingbiofilm formation or disrupting existing or developing biofilms in thesubject. In certain aspects, the composition is a biophotoniccomposition. In certain such aspects, the composition is a topicalbiophotonic composition. In some aspects, the biofilms are formed fromsingle or mixed bacterial species.

In one aspect, this disclosure describes a method for inhibiting biofilmformation or disrupting existing or developing biofilms in a subjectsuffering from venous leg ulcer (VLU), comprising: a) topically applyinga composition comprising at least one chromophore and a pharmaceuticallyacceptable carrier; and b) illuminating said composition with actiniclight, thereby inhibiting biofilm formation or disrupting existing ordeveloping biofilms in the subject. In certain aspects, the compositionis a biophotonic composition. In certain such aspects, the compositionis a topical biophotonic composition. In some aspects, the biofilms areformed from single or mixed bacterial species.

In one aspect, this disclosure describes a method for inhibiting biofilmformation or disrupting existing or developing biofilms in a subjectsuffering from diabetic foot ulcer (DFU), comprising: a) topicallyapplying a composition comprising at least one chromophore and apharmaceutically acceptable carrier; and b) illuminating saidcomposition with actinic light, thereby inhibiting biofilm formation ordisrupting existing or developing biofilms in the subject. In certainaspects, the composition is a biophotonic composition. In certain suchaspects, the composition is a topical biophotonic composition. In someaspects, the biofilms are formed from single or mixed bacterial species.

In one aspect, this disclosure describes a method for inhibiting biofilmformation or disrupting existing or developing biofilms in a subjectsuffering from pressure ulcer (PU), comprising: a) topically applying acomposition comprising at least one chromophore and a pharmaceuticallyacceptable carrier; and b) illuminating said composition with actiniclight, thereby inhibiting biofilm formation or disrupting existing ordeveloping biofilms in the subject. In certain aspects, the compositionis a biophotonic composition. In certain such aspects, the compositionis a topical biophotonic composition. In some aspects, the biofilms areformed from single or mixed bacterial species.

In some embodiments of any of the foregoing or following, saidcomposition is illuminated with actinic light for a period of less thanabout 9 minutes, e.g., for a period of from about 1 second to about 8minutes, from about 1 minute to about 8 minutes, from about 2 minutes toabout 7 minutes, from about 3 minutes to about 6 minutes, from about 4minutes to about 5 minutes. In certain embodiments, said composition isilluminated with actinic light for a period of less than about 5 minutesper cm² of an area to be treated, e.g., for a period of from about 1second to about 5 minutes per cm².

In some embodiments of any of the foregoing or following, the source ofactinic light is placed over an area of biofilm. In some embodiments,said actinic light is visible light having a wavelength between about400 nm and about 700 nm.

In some embodiments of any of the foregoing or following, thechromophore of the composition is chosen from a xanthene derivative dye,an azo dye, a biological stain, and a carotenoid. In certain suchembodiments, said xanthene derivative dye is chosen from a fluorene dye(e.g., a pyronine dye, such as pyronine Y or pyronine B, or a rhodaminedye, such as rhodamine B, rhodamine G, or rhodamine WT), a fluorone dye(e.g., fluorescein, or fluorescein derivatives, such as phloxine B, rosebengal, merbromine, Eosin Y, Eosin B, or Erythrosine B), or a rhodoledye. In certain such embodiments, said azo dye is chosen from methylviolet, neutral red, para red, amaranth, carmoisine, allura red AC,tartrazine, orange G, ponceau 4R, methyl red, and murexide-ammoniumpurpurate. In certain such embodiments, said biological stain is chosenfrom safranin O, basic fuchsin, acid fuschin, 3,3′ dihexylocarbocyanineiodide, carminic acid, and indocyanine green. In certain suchembodiments, said carotenoid is chosen from crocetin, a-crocin(S,S-diapo-S,S-carotenoic acid), zeaxanthine, lycopene, α-carotene,β-carotene, bixin, and fucoxanthine. In certain such embodiments, saidcarotenoid is present in the composition as a mixture chosen fromsaffron red powder, annatto extract, and brown algae extract.

In some embodiments of any of the foregoing or following, thecomposition further comprises at least one oxidant. In certain suchembodiments, the oxidant is chosen from hydrogen peroxide, carbamideperoxide and benzoyl peroxide. In other embodiments, the oxidant ischosen from a peroxy acid and an alkali metal percarbonate.

In some embodiments of any of the foregoing or following, thisdisclosure provides a method for inhibiting biofilm formation ordisrupting existing or developing biofilms in a subject, comprisingtopically applying a composition comprising at least one oxidant andchromophore; and illuminating said composition with actinic light tocause photoillumination of the composition. In certain embodiments, thecomposition is a biophotonic composition. In certain such embodiments,the composition is a topical biophotonic composition.

In some embodiments of any of the foregoing or following, thecomposition further comprises at least one healing factor, e.g.,hyaluronic acid, glucosamine, or allantoin.

In certain aspects of the disclosure, said composition does not comprisethe presence of an oxidant selected from the group consisting of aperoxide, a peroxy acid, hydrogen peroxide, carbamide peroxide, analkali metal peroxide, an alkali metal percarbonate, peroxyacetic acid,and an alkali metal perborate.

In certain embodiments of any of the foregoing or following, thecomposition further comprises one or more salts selected from the groupconsisting of one or more bicarbonate salts, one or more carbonatesalts, and a combination of the foregoing salts.

In certain aspects, the disclosure provides a method for inhibitingbiofilm formation or disrupting existing or developing biofilms in asubject, comprising topically applying a composition, said compositioncomprising at least one chromophore; at least one salt selected from thegroup consisting of one or more bicarbonate salts, one or more carbonatesalts, and a combination of the foregoing salts; and a pharmaceuticallyacceptable carrier; and illuminating said composition to actinic lightto cause photoillumination of the composition. In certain aspects, thecomposition is biophotonic a composition. In certain such aspects, thecomposition is a topical biophotonic composition.

In certain aspects, the disclosure provides a method for inhibitingbiofilm formation or disrupting existing or developing biofilms in asubject suffering from venous leg ulcer (VLU), comprising topicallyapplying a composition, said composition comprising at least onechromophore; at least one salt selected from the group consisting of oneor more bicarbonate salts, one or more carbonate salts, and acombination of the foregoing salts; and a pharmaceutically acceptablecarrier; and illuminating said composition to actinic light to causephotoillumination of the composition. In certain aspects, thecomposition is biophotonic a composition. In certain such aspects, thecomposition is a topical biophotonic composition.

In certain aspects, the disclosure provides a method for inhibitingbiofilm formation or disrupting existing or developing biofilms in asubject suffering from diabetic foot ulcer (DFU), comprising topicallyapplying a composition, said composition comprising at least onechromophore; at least one salt selected from the group consisting of oneor more bicarbonate salts, one or more carbonate salts, and acombination of the foregoing salts; and a pharmaceutically acceptablecarrier; and illuminating said composition to actinic light to causephotoillumination of the composition. In certain aspects, thecomposition is biophotonic a composition. In certain such aspects, thecomposition is a topical biophotonic composition.

In certain aspects, the disclosure provides a method for inhibitingbiofilm formation or disrupting existing or developing biofilms in asubject suffering from pressure ulcer (PU), comprising topicallyapplying a composition, said composition comprising at least onechromophore; at least one salt selected from the group consisting of oneor more bicarbonate salts, one or more carbonate salts, and acombination of the foregoing salts; and a pharmaceutically acceptablecarrier; and illuminating said composition to actinic light to causephotoillumination of the composition. In certain aspects, thecomposition is biophotonic a composition. In certain such aspects, thecomposition is a topical biophotonic composition.

In some embodiments of any of the foregoing or following, thecomposition further comprises at least one gelling agent, e.g., glucose,modified starch, methyl cellulose, carboxymethyl cellulose, propylcellulose, hydroxypropyl cellulose, a carbomer, alginic acid, sodiumalginate, potassium alginate, ammonium alginate, calcium alginate, agar,carrageenan, locust bean gum, pectin, or gelatin.

In certain embodiments of any of the foregoing or following, saidcomposition further comprises at least one buffering agent, such as anethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraaceticacid (EGTA). In certain embodiments of any of the foregoing orfollowing, the chelating agent is EDTA.

In some aspects, there is disclosed a use of a composition, e.g., abiophotonic composition, for the manufacture of a medicament forinhibiting biofilm formation or disrupting existing or developingbiofilms in a subject, wherein the composition comprises at least onechromophore and a pharmaceutically acceptable carrier. In certainaspects, the composition further comprises at least one oxidant. Inother aspects, the composition further comprises one or more saltsselected from the group consisting of one or more bicarbonate salts, oneor more carbonate salts, and a combination of the foregoing salts. Incertain aspects, the composition further comprises at least one healingfactor, e.g., hyaluronic acid, glucosamine, or allantoin.

In certain aspects, there is disclosed a use of a composition e.g., abiophotonic composition, for inhibiting biofilm formation or disruptingexisting or developing biofilms in a subject, wherein the compositioncomprises at least one chromophore and a pharmaceutically acceptablecarrier. In certain aspects, the composition further comprises at leastone oxidant. In other aspects, the composition further comprises one ormore salts selected from the group consisting of one or more bicarbonatesalts, one or more carbonate salts, and a combination of the foregoingsalts. In certain aspects, the composition further comprises at leastone healing factor, e.g., hyaluronic acid, glucosamine, or allantoin.

In certain aspects, there is disclosed a use of a composition e.g., abiophotonic composition, for inhibiting biofilm formation or disruptingexisting or developing biofilms in a subject having VLU, wherein thecomposition comprises at least one chromophore and a pharmaceuticallyacceptable carrier. In certain aspects, the composition furthercomprises at least one oxidant. In other aspects, the compositionfurther comprises one or more salts selected from the group consistingof one or more bicarbonate salts, one or more carbonate salts, and acombination of the foregoing salts. In certain aspects, the compositionfurther comprises at least one healing factor, e.g., hyaluronic acid,glucosamine, or allantoin.

In certain aspects, there is disclosed a use of a composition e.g., abiophotonic composition, for inhibiting biofilm formation or disruptingexisting or developing biofilms in a subject having DFU, wherein thecomposition comprises at least one chromophore and a pharmaceuticallyacceptable carrier. In certain aspects, the composition furthercomprises at least one oxidant. In other aspects, the compositionfurther comprises one or more salts selected from the group consistingof one or more bicarbonate salts, one or more carbonate salts, and acombination of the foregoing salts. In certain aspects, the compositionfurther comprises at least one healing factor, e.g., hyaluronic acid,glucosamine, or allantoin.

In certain aspects, there is disclosed a use of a composition e.g., abiophotonic composition, for inhibiting biofilm formation or disruptingexisting or developing biofilms in a subject having PU, wherein thecomposition comprises at least one chromophore and a pharmaceuticallyacceptable carrier. In certain aspects, the composition furthercomprises at least one oxidant. In other aspects, the compositionfurther comprises one or more salts selected from the group consistingof one or more bicarbonate salts, one or more carbonate salts, and acombination of the foregoing salts. In certain aspects, the compositionfurther comprises at least one healing factor, e.g., hyaluronic acid,glucosamine, or allantoin.

Definitions

Before continuing to describe the present disclosure in further detail,it is to be understood that this disclosure is not limited to specificcompositions or process steps, as such may vary. It must be noted that,as used in this specification and the appended embodiments, the singularform “a”, “an” and “the” include plural referents unless the contextclearly dictates otherwise.

As used herein, the term “about” in the context of a given value orrange refers to a value or range that is within 20%, within 10%, andmore within 5% of the given value or range.

It is convenient to point out here that “and/or” where used herein is tobe taken as specific disclosure of each of the two specified features orcomponents with or without the other. For example “A and/or B” is to betaken as specific disclosure of each of (i) A, (ii) B and (iii) A and B,just as if each is set out individually herein.

The term “biophotonic” as used herein refers to the generation,manipulation, detection and application of photons in a biologicallyrelevant context. In other words, compositions exert their physiologicaleffects primarily due to the generation and manipulation of photons.

The term “composition” is a composition as described herein that may beilluminated with light to induce a production of photons forbiologically relevant applications.

The term “actinic light” is intended to mean light energy emitted from aspecific light source (lamp, LED, or laser) and capable of beingabsorbed by matter (e.g. the chromophore defined below) and produce anidentifiable or measurable change when it interacts with it; asclinically identifiable change we can presume a change in the color ofthe chromophore used (e.g. from red to transparent).

The term “topical” means as applied to body surfaces, such as the skin,mucous membranes, vagina, oral cavity, internal surgical wound sites,and the like.

The term “chromophore” refers to a compound which, when illuminated bylight irradiation, is capable of absorbing the light.

The term “oxidant” as used herein refers to either a compound thatreadily transfers oxygen atoms and oxidizes other compounds, or asubstance that gains electrons in a redox chemical reaction.

The term “chelating agent” as used herein refers to a compound thatbinds metal ions, such as iron, and facilitates their solvation insolution.

The term “healing factor” is intended to mean a compound that promotesor enhances the healing or regenerative process of a tissue.

The term “time of illumination to actinic light” is intended to mean thetime a tissue, skin or wound is illuminated with actinic light perapplication of actinic light.

The term “total time of illumination to actinic light” is intended tomean the cumulative time a tissue, skin or wound is illuminated withactinic light after several application of actinic light.

The term “hydrophilic gelling agent” is intended to mean a material thatthickens and stabilizes liquid solutions, emulsions, and suspensions.Hydrophillic gelling agents dissolve in liquid and provide a structuregiving the resulting gel an appearance of a solid matter, while beingmostly composed of a liquid. Hydrophillic gelling agents are verysimilar to thickeners.

Features and advantages of the subject matter hereof will become moreapparent in light of the following detailed description of selectedembodiments, as illustrated in the accompanying figures. As will berealized, the subject matter disclosed and claimed is capable ofmodifications in various respects, all without departing from the scopeof the claims. Accordingly, the drawings and the description are to beregarded as illustrative in nature, and not as restrictive and the fullscope of the subject matter is set forth in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the present disclosure will becomeapparent from the following detailed description, taken in combinationwith the appended drawings, in which:

FIG. 1 shows results of the mortality of bacteria P. aeruginosa in abiofilm with treatment of a biophotonic composition as disclosed in thisapplication. The P. aeruginosa biofilm was treated with a biophotoniccomposition illuminated in comparison to control composition orillumination alone (illumination in the presence of a compositionlacking both the chromophore and oxidant).

FIG. 2 shows results of the mortality of bacteria S. aureus in a biofilmwith treatment of a biophotonic composition as disclosed in thisapplication. The S. aureus biofilm was treated with a biophotoniccomposition illuminated in comparison to control composition orillumination alone (illumination in the presence of a compositionlacking both the chromophore/oxidant).

DETAILED DESCRIPTION

In one aspect, this disclosure describes a method for inhibiting biofilmformation or disrupting existing or developing biofilms in a subject(e.g., a subject suffering from VLU, DFU, and/or PU), comprising: a)topically applying a composition comprising at least one chromophore anda pharmaceutically acceptable carrier; and b) illuminating saidcomposition to actinic light to cause photoillumination of thecomposition. In certain embodiments, the composition is a biophotoniccomposition. In certain such embodiments, the composition is a topicalbiophotonic composition.

In certain embodiments of any of the foregoing or following, thechromophore is selected from the group consisting of a xanthenederivative dye, an azo dye, a biological stain, and a carotenoid. Incertain such embodiments, the at least one chromophore is selected fromthe group consisting of eosin (e.g., eosin B or eosin Y), erythrosine(e.g., erythrosine B), and Saffron red powder.

In certain embodiments of any of the foregoing or following, thecomposition comprises at least one chromophore present in an amount ofat least about 0.2% by weight of the composition, e.g., from about 0.02%to about 12%, from 0.02% to about 10%, from 0.02% to about 8%, fromabout 0.02% to about 6%, from about 0.02% to about 4%, from about 0.02%to about 2%, from about 0.02% to about 1%, or about 0.5% by weight ofthe composition.

In some embodiments of any of the foregoing or following, thecomposition further comprises an additional chromophore selected fromthe group consisting of Xanthene derivative dye, azo dye, biologicalstain, and carotenoid.

In some embodiments of any of the foregoing or following, thechromophore or chromophore of the composition is selected from the groupconsisting of a xanthene derivative dye, an azo dye, a biological stain,and a carotenoid. In certain such embodiments, said xanthene derivativedye is chosen from a fluorene dye (e.g., a pyronine dye, such aspyronine Y or pyronine B, or a rhodamine dye, such as rhodamine B,rhodamine G, or rhodamine WT), a fluorone dye (e.g., fluorescein, orfluorescein derivatives, such as phloxine B, rose bengal, merbromine,Eosin Y, Eosin B, or Erythrosine B), or a rhodole dye. In certain suchembodiments, said azo dye is chosen from methyl violet, neutral red,para red, amaranth, carmoisine, allura red AC, tartrazine, orange G,ponceau 4R, methyl red, and murexide-ammonium purpurate. In certain suchembodiments, said biological stain is chosen from safranin O, basicfuchsin, acid fuschin, 3,3′ dihexylocarbocyanine iodide, carminic acid,and indocyanine green. In certain such embodiments, said carotenoid ischosen from crocetin, a-crocin (S,S-diapo-S,S-carotenoic acid),zeaxanthine, lycopene, α-carotene, β-carotene, bixin, and fucoxanthine.In certain such embodiments, said carotenoid is present in thecomposition as a mixture is selected from the group consisting ofsaffron red powder, annatto extract, and brown algae extract.

In some embodiments of any of the foregoing or following, the additionalchromophore is selected from a group consisting of phloxine B, rosebengal, eosin B, fluorescein, erythrosine B, rhodamine B, rhodamine G,rhodamine WT, saffron red powder, annatto extract, brown algae extract,safranin O, basic fuchsin, acid fuschin, 3,3′ dihexylocarbocyanineiodide, carminic acid, indocyanine green, crocetin, α-crocin(8,8-diapo-8,8-carotenoic acid), zeaxanthine, lycopene, α-carotene,β-carotene, bixin, fucoxanthine, methyl violet, neutral red, para red,amaranth, carmoisine, allura red AC, tartrazine, orange G, ponceau 4R,methyl red, murexide-ammonium purpurate, pyronine Y and pyronine B.

In certain embodiments of any of the foregoing or following, theadditional chromophore is present in an amount of from about 0.02% toabout 12% by weight of the composition, such as from about 0.02% toabout 10%, from about 0.02% to about 8%, from about 0.02% to about 6%,from about 0.02% to about 4%, from about 0.02% to about 2%, from about0.02% to about 1%, or about 5% by weight of the composition.

In some embodiments of any of the foregoing or following, thecomposition further comprises at least one oxidant. In certain suchembodiments, the oxidant is selected from the group consisting ofhydrogen peroxide, carbamide peroxide, and benzoyl peroxide. In otherembodiments, the oxidant is a peroxy acid or an alkali metalpercarbonate.

In some embodiments of any of the foregoing or following, the oxidant ispresent in an amount of from about 1% to about 70% by weight of thecomposition, e.g., from about 1% to about 60%, from about 1% to about50%, from about 1% to about 40%, from about 1% to about 30%, from about1% to about 20%, from about 1% to about 18%, from about 1% to about 16%,from about 1% to about 14%, from about 1% to about 12%, from about 1% toabout 10%, from about 1% to about 8%, from about 1% to about 6%, fromabout 0.05% to about 6%, from about 0.1% to about 6%, from about 0.5% toabout 6%, from about 2.5% to about 6%, from about 3.5% to about 6% byweight of the composition.

In some embodiments of any of the foregoing or following, the oxidant ishydrogen peroxide and is present in an amount of from about 3.5% toabout 6% by weight of the composition.

In some embodiments of any of the foregoing or following, the oxidant iscarbamide peroxide and is present in an amount of from about 10% toabout 16% by weight of the composition.

In some embodiments of any of the foregoing or following, the oxidant isbenzoyl peroxide and is present in an amount of from about 2.5% to about5% by weight of the composition.

In some embodiments of any of the foregoing or following, thisdisclosure provides a method of inhibiting biofilm formation ordisrupting existing or developing biofilm in a subject, comprisingtopically applying a composition comprising at least one oxidant and atleast one chromophore, and illuminating said composition to actiniclight to cause photoillumination of the composition. In certain suchembodiments, the composition is a biophotonic composition.

In some embodiments of any of the foregoing or following, saidcomposition does not comprise an oxidant, such as an oxidant selectedfrom the group consisting of a peroxide, a peroxy acid, hydrogenperoxide, carbamide peroxide, an alkali metal peroxide, an alkali metalpercarbonate, peroxyacetic acid, and an alkali metal perborate.

In some embodiments of any of the foregoing or following, thecomposition further comprises at least one salt selected from the groupconsisting of one or more bicarbonate salts, one or more carbonatesalts, and a combination of the foregoing salts.

In some embodiments of any of the foregoing or following, the at leastone bicarbonate salt is selected from the group consisting of ammoniumbicarbonate, caesium bicarbonate, potassium bicarbonate, sodiumbicarbonate, choline bicarbonate, aminoguanidine bicarbonate, andtetraethylammonium bicarbonate. In certain such embodiments, thebicarbonate salt is sodium bicarbonate or potassium bicarbonate.

In some embodiments of any of the foregoing or following, the at leastone carbonate salt is selected from the group consisting of bariumcarbonate, beryllium carbonate, caesium carbonate, calcium carbonate,cobalt (II) carbonate, copper (II) carbonate, lithium carbonate,magnesium carbonate, nickel (II) carbonate, potassium carbonate, sodiumcarbonate, and zinc carbonate. In certain such embodiments, thecarbonate salt is selected from the group consisting of sodiumcarbonate, calcium carbonate, and potassium bicarbonate.

In some embodiments of any of the foregoing or following, the disclosureprovides a method of inhibiting biofilm formation or disrupting existingor developing biofilm in a subject (e.g., suffering from DFU, VLU,and/or PU), comprising topically applying a composition comprising atleast one chromophore; at least one salt, such as a salt selected fromthe group consisting of one or more bicarbonate salts, one or morecarbonate salts, and a combination of the foregoing salts; and apharmaceutically acceptable carrier; and illuminating said compositionto actinic light to cause photoillumination of the composition. Incertain such embodiments, the composition is a biophotonic composition.

In some embodiments of any of the foregoing or following, thecomposition further comprises at least one healing factor, such ashyaluronic acid, glucosamine, or allantoin.

In certain embodiments of any of the foregoing or following, saidcomposition further comprises at least one buffering agent chosen fromethylenediaminetetraacetic acid (EDTA) and ethylene glycol tetraaceticacid (EGTA). In certain embodiments of any of the foregoing orfollowing, the chelating agent is EDTA.

In certain embodiments of any of the foregoing or following, thecomposition further comprises at least one hydrophilic gelling agent. Incertain such embodiments, the hydrophilic gelling agent is selected fromthe group consisting of glucose, modified starch, methyl cellulose,carboxymethyl cellulose, propyl cellulose, hydroxypropyl cellulose,Carbopol® polymers, alginic acid, sodium alginate, potassium alginate,ammonium alginate, calcium alginate, agar, carrageenan, locust bean gum,pectin, and gelatin. The hydrophilic gelling agent enhances theconsistency of the composition and contributes to facilitating theapplication of the composition to the skin or area of biofilms.

In some embodiments of any of the foregoing or following, saidcomposition is illuminated with actinic light, such as a LED light, forat least one treatment period of from about 1 minute to about 30 minutesper cm² of an area to be treated. In certain such embodiments, saidcomposition is illuminated with actinic light for at least one treatmentperiod of from about 4 minutes to about 26 minutes, from about 8 minutesto about 24 minutes, from about 10 minutes to about 20 minutes, fromabout 10 minutes to about 18 minutes; or from about 1 minutes to about 5minutes, from about 5 minutes to about 10 minutes, from about 10 minutesto about 15 minutes, from about 15 minutes to about 20 minutes, fromabout 20 minutes to about 25 minutes, or about 5 minutes, about 10minutes, about 15 minutes, or about 20 minutes per cm² of an area to betreated, such as an area of biofilms.

In some embodiments of any of the foregoing or following, saidcomposition is illuminated with actinic light for at least one treatmentperiod of from about 1 minute to about 9 minutes per cm² of an area tobe treated. In certain such embodiments, said composition is illuminatedwith actinic light for at least one treatment period of from about 2minutes to about 8 minutes, from about 3 minutes to about 7 minutes,from about 4 minutes to about 6 minutes, or from about 5 minutes per cm²of an area to be treated.

In some embodiments of any of the foregoing or following, saidcomposition is illuminated with actinic light for at least two treatmentperiods (e.g., two consecutive treatment periods). In certain suchembodiments, said composition is illuminated with actinic light for atleast two treatment periods, each treatment period followed by a restinginterval.

In some embodiments of any of the foregoing or following, the restinginterval is from about 1 minute to about 30 minutes, such as from about4 minutes to about 26 minutes, from about 8 minutes to about 24 minutes,from about 10 minutes to about 20 minutes, from about 10 minutes toabout 18 minutes; or from about 1 minutes to about 5 minutes, from about1 minute to about 2 minutes, from about 1 minute to about 3 minutes,from about 2 minutes to about 8 minutes, from about 3 minutes to about 7minutes, from about 4 minutes to about 6 minutes, from about 5 minutesto about 10 minutes, from about 5 minutes to about 10 minutes, fromabout 10 minutes to about 15 minutes, from about 15 minutes to about 20minutes, from about 20 minutes to about 25 minutes, or about 5 minutes,about 10 minutes, about 15 minutes, or about 20 minutes per cm² of anarea to be treated, such as an area of biofilms.

In some embodiments of any of the foregoing or following, saidcomposition is illuminated with at least two treatment periods (e.g.,two consecutive treatment periods) of actinic light wherein eachtreatment period is from about 1 minute to about 10 minutes per cm² ofan area to be treated (e.g., e.g., from about 2 minutes to about 8minutes, from about 3 minutes to about 7 minutes, from about 4 minutesto about 6 minutes, or about 5 minutes), wherein each treatment periodis followed by a resting interval for from about 1 minute to about 10minutes (e.g., from about 2 minutes to about 8 minutes, from about 3minutes to about 7 minutes, from about 4 minutes to about 6 minutes,from about 1 minute to about 2 minutes, from about 1 minute to about 3minutes, from about 5 minutes to about 10 minutes, or about 5 minutes).

In some embodiments of any of the foregoing or following, saidcomposition is illuminated with at least two treatment periods (e.g.,two consecutive treatment periods) of actinic light wherein eachtreatment period is from about 1 minute to about 5 minutes per cm² of anarea to be treated, wherein each treatment period is followed by aresting interval for from about 1 minute to about 10 minutes (e.g., fromabout 2 minutes to about 8 minutes, from about 3 minutes to about 7minutes, from about 4 minutes to about 6 minutes, from about 1 minute toabout 2 minutes, from about 1 minute to about 3 minutes, from about 5minutes to about 10 minutes, or about 5 minutes).

In some embodiments of any of the foregoing or following, saidcomposition is illuminated with at least two treatment periods (e.g.,two consecutive treatment periods) of actinic light wherein eachtreatment period is from about 1 minute to about 5 minutes per cm² of anarea to be treated, wherein each treatment period is followed by aresting interval for from about 5 minutes.

In some embodiments of any of the foregoing or following, said methodfurther comprising:

a) topically applying the composition to the subject's area of biofilm;

b) illuminating the subject's area of biofilm to actinic light for atreatment period of from about 1 minute to about 10 minutes (e.g., fromabout 1 minute to 5 minutes or about 5 minutes);

c) removing the source of actinic light away from the subject's area ofbiofilm for a resting interval of from about 1 minute to about 10minutes (e.g., from about 1 minute to 5 minutes, or about 5 minutes);

d) illuminating the subject's area of biofilm to actinic light for asecond treatment period of from about 1 minute to about 10 minutes(e.g., from about 1 minute to 5 minutes, or about 5 minutes); andwherein the first illumination to actinic light activates thecomposition.

In some embodiments of any of the foregoing or following, said methodfurther comprising:

a) topically applying the composition to the subject's area of biofilm;

b) illuminating the subject's area of biofilm to actinic light for atreatment period of from about 1 minute to about 10 minutes (e.g., fromabout 1 minute to 5 minutes or about 5 minutes);

c) removing the source of actinic light away from the subject's area ofbiofilm for a resting interval of from about 1 minute to about 5minutes;

d) illuminating the subject's area of biofilm to actinic light for asecond treatment period of from about 1 minute to about 10 minutes(e.g., from about 1 minute to 5 minutes or about 5 minutes); and whereinthe first illumination to actinic light activates the composition.

In some embodiments of any of the foregoing or following, the methodfurther comprises topically re-applying the composition before eachtreatment period, e.g., before the second treatment period.

In some embodiments of any of the foregoing or following, the source ofactinic light is illuminating in continuous motion over an area to betreated.

In some embodiments of any of the foregoing or following, the source ofactinic light is positioned over an area to be treated. In someembodiments, said actinic light is visible light having a wavelengthbetween about 400 nm and about 700 nm.

In some embodiments of any of the foregoing or following, the subject isa mammal, such as a human, an equine, a feline, or a canine.

In some embodiments of any of the foregoing or following, the biofilm isassociated with the subject's skin. In other embodiments, the biofilm isassociated with the subject's soft tissues.

In some embodiments of any of the foregoing or following, the biofilmcomprises Staphylococcus aureus.

In some embodiments of any of the foregoing or following, the biofilmcomprises Pseudomonas aeruginosa.

In some embodiments of any of the foregoing or following, the biofilmcomprises Staphylococcus aureus and Pseudomonas aeruginosa.

In some embodiments of any of the foregoing or following, the biofilmcomprises gram-negative bacteria. In other embodiments, the biofilmcomprises gram-positive bacteria.

In some embodiments of any of the foregoing or following, the biofilm isformed by a microorganism.

In some embodiments of any of the foregoing or following, the biofilmcomprises a polysaccharide, protein, or glycopeptide produced by amicroorganism.

In some embodiments of any of the foregoing or following, the biofilm isproduced by bacteria. In other embodiments, the biofilm is produced byyeast. In other embodiments, the biofilm is produced by fungi.

In some embodiments of any of the foregoing or following, the subject isa mammal, such as a human, an equine, a feline, or a canine.

In some aspects, the disclosure provides for use of a composition forthe manufacture of a medicament for inhibiting biofilm formation ordisrupting existing or developing biofilms in a subject (e.g., sufferingfrom VLU, DFU, and/or PU), wherein said composition comprises at leastone chromophore and a pharmaceutically acceptable carrier. In certainaspects, the composition further comprises at least one oxidant. Inother aspects, the composition further comprises one or more saltsselected from the group consisting of one or more bicarbonate salts, oneor more carbonate salts, and a combination of the foregoing salts. Incertain aspects, the composition further comprises at least one healingfactor, e.g., hyaluronic acid, glucosamine, or allantoin.

In some aspects, the disclosure provides for use of a composition forinhibiting biofilm formation or disrupting existing or developingbiofilms in a subject (e.g., suffering from VLU, DFU, and/or PU), thecomposition comprising: at least one chromophore and a pharmaceuticallyacceptable carrier. In certain such aspects, the composition furthercomprises at least one oxidant. In other aspects, the compositionfurther comprises one or more salts selected from the group consistingof one or more bicarbonate salts, one or more carbonate salts, and acombination of the foregoing salts. In certain aspects, the compositionfurther comprises at least one healing factor, e.g., hyaluronic acid,glucosamine, or allantoin.

Compositions

The present disclosure provides methods and uses comprising acomposition, e.g., a composition for inhibiting biofilm formation ordisrupting existing or developing biofilms in a subject. In one aspect,the composition of the present disclosure is a biophotonic composition.Compositions of this disclosure, in a broad sense, are activated bylight (e.g., photons) of specific wavelength. These compositions containat least one exogenous chromophore which is capable of absorbing lightand accelerates the dispersion of light energy.

When a chromophore absorbs a photon of a certain wavelength, it becomesexcited. This is an unstable condition and the molecule tries to returnto the ground state, giving away the excess energy. For somechromophores, it is favorable to emit the excess energy as light whentransforming back to the ground state. This process is calledfluorescence. The peak wavelength of the emitted fluorescence is shiftedtowards longer wavelengths compared to the absorption wavelengths(‘Stokes’ shift’). The emitted fluorescent energy can then betransferred to the other components of the composition or to a treatmentsite on to which the composition is topically applied. Differingwavelengths of light may have different and complementary therapeuticeffects on tissue.

In certain embodiments, the compositions of the present disclosure aresubstantially transparent/translucent and/or have high lighttransmittance in order to permit light dissipation into and through thecomposition.

The % transmittance of the composition can be measured in the range ofwavelengths from 250 nm to 800 nm using, for example, a Perkin-ElmerLambda 9500 series UV-visible spectrophotometer. Alternatively, aSynergy HT spectrophotometer (BioTek Instrument, Inc.) can be used inthe range of wavelengths from 380 nm to 900 nm.

Transmittance is calculated according to the following equation:

$A_{\lambda} = {{\log_{10}\frac{I_{0}}{I}} = {\log_{10}{\frac{1}{T}.}}}$

where A is absorbance, T is transmittance, I₀ is intensity of radiationbefore passing through material, and I is intensity of light passingthrough material.

The values can be normalized for thickness. As stated herein, %transmittance (translucency) is as measured for a 2 mm thick sample at awavelength of 526 nm. It will be clear that other wavelengths can beused.

In certain embodiments of the disclosure, the compositions of thepresent disclosure are for topical uses. The composition can be in theform of a semi-solid or viscous liquid, such as a gel, or are gel-like,and which have a spreadable consistency at room temperature (e.g., about20-25° C.) prior to illumination. In certain such embodiments whereinthe composition has a spreadable consistency, the composition can betopically applied to a treatment site at a thickness of from about 0.5mm to about 3 mm, from about 0.5 mm to about 2.5 mm, or from about 1 mmto about 2 mm. In some embodiments, the composition can be topicallyapplied to a treatment site at a thickness of about 2 mm or about 1 mm.Spreadable compositions can conform to a topography of a treatment site.This can have advantages over a non-conforming material in that a betterand/or more complete illumination of the treatment site can be achievedand the compositions are easy to apply and remove.

These compositions may be described based on the components making upthe composition. Additionally or alternatively, the compositions of thepresent disclosure have functional and structural properties and theseproperties may also be used to define and describe the compositions.Individual components of the composition of the present disclosure aredetailed as below.

Oxidants

In certain embodiments, the composition of the present disclosure maycontain an oxidant. In certain embodiments, the oxidant is a peroxidecompound; peroxide compounds are oxidants that contain the peroxy group(R—O—O—R), which is a chainlike structure containing two oxygen atoms,each of which is bonded to the other and a radical or some element.Suitable oxidants for preparation of the active medium include, but arenot limited to:

Hydrogen peroxide (H₂O₂) is the starting material to prepare organicperoxides. H₂O₂ is a powerful oxidizing agent, and the unique propertyof hydrogen peroxide is that it breaks down into water and oxygen anddoes not form any persistent, toxic residual compound. Hydrogen peroxidefor use in this composition can be used in a gel, for example with 6%hydrogen peroxide. A suitable range of concentration over which hydrogenperoxide can be used in the present composition is less than about 12%,or from about 1% to about 12%, preferably from about 3.5% to about 12%and most preferably from about 3.5% to about 6%.

Urea hydrogen peroxide (also known as urea peroxide, carbamide peroxideor percarbamide) is soluble in water and contains approximately 36%hydrogen peroxide. Carbamide peroxide for use in this composition can beused as a gel, for example with 16% carbamide peroxide that representsapproximately 5.6% hydrogen peroxide. A suitable range of concentrationover which urea peroxide can be used in the present composition is lessthan 36%, or from about 3% to about 36%, and preferably from about 10%to about 36% and most preferably from about 3% to about 16%. Ureaperoxide breaks down to urea and hydrogen peroxide in a slow-releasefashion that can be accelerated with heat or photochemical reactions.The released urea [carbamide, (NH₂)CO₂)], is highly soluble in water andis a powerful protein denaturant. It increases solubility of someproteins and enhances rehydration of the skin and/or mucosa.

Benzoyl peroxide consists of two benzoyl groups (benzoic acid with the Hof the carboxylic acid removed) joined by a peroxide group. It is foundin treatments for acne, in concentrations varying from 2.5% to 10%. Thereleased peroxide groups are effective at killing bacteria. Benzoylperoxide also promotes skin turnover and clearing of pores, whichfurther contributes to decreasing bacterial counts and reduce acne.Benzoyl peroxide breaks down to benzoic acid and oxygen upon contactwith skin, neither of which are toxic. A suitable range of concentrationover which benzoyl peroxide can be used in the present composition isless than about 10%, or from about 1% to about 10%, or preferably fromabout 1% to about 8%, and most preferably from about 2.5% to about 5%.

Suitable oxidants may also include peroxy acids and alkali metalpercarbonates, but the inclusion of any other forms of peroxides (e.g.organic or inorganic peroxides) should be avoided due to their increasedtoxicity and their unpredictable reaction with the photodynamic energytransfer.

Chromophores

The compositions, such as biophotonic topical compositions, the methodsand uses of the present disclosure comprise one or more chromophores,which can be considered exogenous, e.g., are not naturally present inskin or tissue. When a composition of the present disclosure isilluminated with light, the chromophore(s) are excited to a higherenergy state. When the chromophore(s)' electrons return to a lowerenergy state, they emit photons with a lower energy level, thus causingthe emission of light of a longer wavelength (Stokes' shift).

Suitable chromophores for the compositions of the disclosure can befluorescent dyes (or stains), although other dye groups or dyes(biological and histological dyes, food colorings, carotenoids,naturally occurring fluorescent and other dyes) can also be used.

In some embodiments, the composition of the present disclosure comprisesa chromophore which undergoes partial or complete photobleaching uponapplication of light. By photobleaching is meant a photochemicaldestruction of the chromophore which can generally be visualized as aloss of color.

In some embodiments, the chromophore absorbs at a wavelength in therange of the visible spectrum, such as at a wavelength of from about380-800 nm, about 380-700 nm, or about 380-600 nm. In some embodiments,the chromophore absorbs at a wavelength of about 200-800 nm, about200-700 nm, about 200-600 nm or about 200-500 nm. In some embodiments,the chromophore absorbs at a wavelength of about 200-600 nm. In someembodiments, the chromophore absorbs light at a wavelength of about200-300 nm, about 250-350 nm, about 300-400 nm, about 350-450 nm, about400-500 nm, about 400-600 nm, about 450-650 nm, about 600-700 nm, about650-750 nm or about 700-800 nm.

In some embodiments, the chromophore or combination of chromophores ispresent in an amount of about 0.001-40% by weight of the composition. Insome embodiments, the chromophore or combination of chromophores ispresent in an amount of about 0.005-2%, about 0.01-1%, about 0.01-2%,about 0.05-1%, about 0.05-2%, about 0.1-1%, about 0.1-2%, about 1-5%,about 2.5-7.5%, about 5-10%, about 7.5-12.5%, about 10-15%, about12.5-17.5%, about 15-20%, about 17.5-22.5%, about 20-25%, about22.5-27.5%, about 25-30%, about 27.5-32.5%, about 30-35%, about32.5-37.5%, or about 35-40% by weight of the composition. In someembodiments, the chromophore or combination of chromophores is presentin an amount of at least about 0.2% by weight of the composition.

In some embodiments, the chromophore or combination of chromophores ispresent in an amount of from 0.001-40% by weight of the composition. Insome embodiments, the chromophore or combination of chromophores ispresent in an amount of 0.005-2%, 0.01-1%, 0.01-2%, 0.05-1%, 0.05-2%,0.1-1%, 0.1-2%, 1-5%, 2.5-7.5%, 5-10%, 7.5-12.5%, 10-15%, 12.5-17.5%,15-20%, 17.5-22.5%, 20-25%, 22.5-27.5%, 25-30%, 27.5-32.5%, 30-35%,32.5-37.5%, or 35-40% by weight of the composition. In some embodiments,the chromophore or combination of chromophores is present in an amountof at least 0.2% by weight of the composition.

It will be appreciated to those skilled in the art that opticalproperties of a particular chromophore may vary depending on thechromophore's surrounding medium. Therefore, as used herein, aparticular chromophore's absorption and/or emission wavelength (orspectrum) corresponds to the wavelengths (or spectra) measured in acomposition of the present disclosure.

The compositions disclosed herein may include at least one additionalchromophore. Combining chromophores may increase photo-absorption by thecombined dye molecules and enhance absorption and photo-biomodulationselectivity. This creates multiple possibilities of generating newphotosensitive, and/or selective chromophores mixtures.

When such multi-chromophore compositions are illuminated with light,energy transfer can occur between the chromophores. This process, knownas resonance energy transfer, is a photophysical process through whichan excited ‘donor’ chromophore (also referred to herein as firstchromophore) transfers its excitation energy to an ‘acceptor’chromophore (also referred to herein as second chromophore). Theefficiency and directedness of resonance energy transfer depends on thespectral features of donor and acceptor chromophores. In particular, theflow of energy between chromophores is dependent on a spectral overlapreflecting the relative positioning and shapes of the absorption andemission spectra. For energy transfer to occur the emission spectrum ofthe donor chromophore overlap with the absorption spectrum of theacceptor chromophore.

Energy transfer manifests itself through decrease or quenching of thedonor emission and a reduction of excited state lifetime accompaniedalso by an increase in acceptor emission intensity.

To enhance the energy transfer efficiency, the donor chromophore shouldhave good abilities to absorb photons and emit photons. Furthermore, itis thought that the more overlap there is between the donorchromophore's emission spectra and the acceptor chromophore's absorptionspectra, the better a donor chromophore can transfer energy to theacceptor chromophore.

In some embodiments, the biophotonic topical composition of the presentdisclosure further comprises an acceptor, or a second, chromophore. Insome embodiments, the donor, or first, chromophore has an emissionspectrum that overlaps at least about 80%, about 70%, about 60%, about50%, about 40%, about 30%, about 20%, or about 10% with an absorptionspectrum of the second chromophore. In some embodiments, the firstchromophore has an emission spectrum that overlaps at least about 20%with an absorption spectrum of the second chromophore. In someembodiments, the first chromophore has an emission spectrum thatoverlaps at least 1-10%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%,35-45%, 50-60%, 55-65% or 60-70% with an absorption spectrum of thesecond chromophore.

% spectral overlap, as used herein, refers to the % overlap of a donorchromophore's emission wavelength range with an acceptor chromophore'sabsorption wavelength range, measured at spectral full width quartermaximum (FWQM). The spectral FWQM of the acceptor chromophore'sabsorption spectrum is from about 60 nm (about 515 nm to about 575 nm).The overlap of the donor chromophore's spectrum with the absorptionspectrum of the acceptor chromophore is about 40 nm (from 515 nm toabout 555 nm). Thus, the % overlap can be calculated as 40 nm/60nm×100=66.6%.

In some embodiments, the second chromophore absorbs at a wavelength inthe range of the visible spectrum, e.g., 400-700 nm. In someembodiments, the second chromophore has an absorption wavelength that isrelatively longer than that of the first chromophore within the range ofabout 50-250 nm, about 25-150 nm or about 10-100 nm.

As discussed above, the application of light to the compositions of thepresent disclosure can result in a cascade of energy transfer betweenthe chromophores. In some embodiments, such a cascade of energy transferprovides photons that penetrate the epidermis, dermis and/or mucosa atthe target tissue, including, such as, a site of wound.

In some embodiments, the chromophore or chromophores are selected suchthat their emitted fluorescent light, on photoillumination, is withinone or more of the green, yellow, orange, red and infrared portions ofthe electromagnetic spectrum, for example having a peak wavelengthwithin the range of about 490 nm to about 800 nm. In some embodiments,the emitted fluorescent light has a power density of between 0.005 toabout 10 mW/cm², about 0.5 to about 5 mW/cm².

Suitable chromophores useful in the compositions (such as thebiophotonic compositions), methods, and uses of the present disclosureinclude, but are not limited to the following:

Xanthene Derivatives

The xanthene derivative dyes have been used and tested for a long timeworldwide. They display low toxicity and increased fluorescence. Thexanthene group consists of 3 sub-groups that are: a) the fluorenes; b)fluorones; and c) the rhodoles.

The fluorenes group comprises the pyronines (e.g. pyronin Y and B) andthe rhodamines (e.g. rhodamine B, G and WT). Depending on theconcentration used, both pyronines and rhodamines may be toxic and theirinteraction with light may lead to increased toxicity. Similar effectsare known to occur for the rhodole dye group.

The fluorone group comprises the fluorescein dye and the fluoresceinderivatives.

Fluorescein is a fluorophore commonly used in microscopy with anabsorption max. of 494 nm and an emission max. of 521 nm. The disodiumsalt of fluorescein is known as D&C Yellow 8. It has very highfluorescence but photodegrades quickly. In the present composition,mixtures of fluorescein with other chromophores such as indocyanin greenand/or saffron red powder will confer increased photoadsorption to theseother compounds.

Eosins group comprises eosin Y (tetrabromofluorescein, acid red 87, D&CRed 22) with an abs. max 514-518 nm, stains cytoplasm of cells,collagen, muscle fibers and red blood cells intensely red; and eosin B(acid red 91, eosin scarlet, dibromo-dinitrofluorescein), with the samestaining characteristics as eosin Y. Eosin Y and eosin B arecollectively referred to as “Eosin”, and use of the term “Eosin” refersto either eosin Y, eosin B or a mixture of both. Eosin Y, eosin B, or amixture of both can be used because of their sensitivity to the lightspectra used: broad spectrum blue light, blue to green light and greenlight. Their tissue and biofilm staining properties and their lowtoxicity are also advantageous. Both eosin Y and eosin B stain red bloodcells and thus confer to the composition of the present disclosurehaemostatic (controls the flow or stops the flow of blood) properties aswell as increase the selective targeting of light to the soft tissues ofthe lesion or wound during the application of the composition. Inembodiments, the composition includes in the range of less than about12% of at least one of eosin B or eosin Y or combinations thereof, orfrom about 0.02% to about 12% of at least one of eosin B or eosin Y orcombinations thereof, or between about 0.02% and about 1.2%, or fromabout 0.02% to about 0.5%, or from about 0.5% to about 0.8% of at leastone of eosin B or eosin Y or combinations thereof. In yet anotherembodiment, the composition includes less than 12% of at least one ofeosin B or eosin Y or combinations thereof, or from about 0.02% to about12% of at least one of eosin B or eosin Y or combinations thereof, orbetween about 0.02% and about 1.2%, or from about 0.02% to about 0.5%,or from about 0.02% to about less than 0.5% or from about 0.5% to about0.8% of at least one of eosin B or eosin Y or combinations thereof,and/or less than about 2% erythrosine B, or from about 0.005 to about 2%erythrosine B, or from about 0.005% to about 1%, or about 0.01% to about1%, or about 0.005% and about 0.15% of erythrosine B.

Phloxine B (2,4,5,7-tetrabromo 4,5,6,7,tetrachlorofluorescein, D&C Red28, acid red 92) is a red dye derivative of fluorescein which is usedfor disinfection and detoxification of waste water throughphotooxidation. It has an abs. max. of 535-548 nm. It is also used as anintermediate for making photosensitive dyes and drugs.

Erythrosine B, or simply Erythrosine (acid red 51, tetraiodofluorescein)is a cherry-pink, coal-based fluorine food dye used as a biologicalstain, and a biofilm and dental plaque disclosing agent, with max. abs.524-530 nm in aqueous solution. It is subject to photodegradation.Erythrosine is also used in some embodiments due to its photosensitivityto the light spectra used and its ability to stain biofilms. Inembodiments, the composition includes in the range of less than about 2%erythrosine B, or from about 0.005 to about 2% erythrosine B, or fromabout 0.005% to about 1%, or about 0.01% to about 1%, or about 0.005%and about 0.15% of erythrosine B.

Rose Bengal (4,5,6,7 tetrachloro 2,4,5,7 tetraiodofluorescein, acid red94) is a bright bluish-pink biological dye with an absorption max. of544-549 nm, that has been used as a dye, biological stain and diagnosticaid. Also it is used in synthetic chemistry to generate singlet fromtriplet oxygen.

Merbromine (mercurochrome) is an organo-mercuric disodium salt offluorescein with an abs. max. of 508 nm. It is used as an antiseptic.

Azo Dyes

The azo (or diazo-) dyes share the N—N group, called azo the group. Theyare used mainly in analytical chemistry or as food colorings and are notfluorescent. Suitable azo dyes include: Methyl violet, neutral red, parared (pigment red 1), amaranth (Azorubine S), Carmoisine (azorubine, foodred 3, acid red 14), allura red AC (FD&C 40), tartrazine (FD&C Yellow5), orange G (acid orange 10), Ponceau 4R (food red 7), methyl red (acidred 2), murexide-ammonium purpurate.

Biological Stains

Dye molecules commonly used in staining protocols for biologicalmaterials can also be used as chromophores. Suitable biological stainsinclude:

Saffranin (Saffranin 0, basic red 2) is also an azo-dye and is used inhistology and cytology. It is a classic counter stain in a Gram stainprotocol.

Fuchsin (basic or acid) (rosaniline hydrochloride) is a magentabiological dye that can stain bacteria and has been used as anantiseptic. It has an abs. max. 540-555 nm.

3,3′ dihexylocarbocyanine iodide (DiOC6) is a fluorescent dye used forstaining cell's endoplasmic reticulum, vesicle membranes andmitochondria. It shows photodynamic toxicity; when illuminated with bluelight, has a green fluorescence.

Carminic acid (acid red 4, natural red 4) is a red glucosidalhydroxyanthrapurin naturally obtained from cochineal insects.

Indocyanin green (ICG) is used as a diagnostic aid for blood volumedetermination, cardiac output, or hepatic function. ICG binds stronglyto red blood cells and when used in mixture with fluorescein, itincreases the absorption of blue to green light.

Carotenoids

Carotenoid dyes can also act as chromophores.

Saffron red powder is a natural carotenoid-containing compound. Saffronis a spice derived from Crocus sativus. It is characterized by a bittertaste and iodoform or hay-like fragrance; these are caused by thecompounds picrocrocin and saffranal. It also contains the carotenoid dyecrocin that gives its characteristic yellow-red color.

Saffron contains more than 150 different compounds many of them arecarotenoids: mangicrocin, reaxanthine, Iycopene, and various α andβ-carotenes, which show good absorption of light and beneficialbiological activity. Also saffron can act as both a photon-transferagent and a healing factor. Saffron color is primarily the result ofα-crocin (8,8 diapo-8,8-carotenoid acid). Dry saffron red powder ishighly sensitive to fluctuating pH levels and rapidly breaks downchemically in the presence of light and oxidizing agents. It is moreresistant to heat. Data show that saffron has anticarcinogenic,immunomodulating and antioxidant properties. For absorbance, it isdetermined for the crocin specific photon wavelength of 440 nm (bluelight). It has a deep red colour and forms crystals with a melting pointof 186° C. When dissolved in water it forms an orange solution.

Crocetin is another compound of saffron that was found to express anantilipidemic action and promote oxygen penetration in differenttissues. More specifically it was observed an increased oxygenation ofthe endothelial cells of the capillaries. An increase of the oxygenationof muscles and cerebral cortex was observed and led to an increasedsurvival rate in laboratory animals with induced hemorrhagic shock oremphysema.

Anatto is a spice that contains as its main constituent (70-80%) thecarotenoid bixin which displayed relevant antioxidative properties.

β-carotene, also displayed suitable characteristics.

Fucoxanthine is a constituent of brown algae with a pronounced abilityfor photosensitization of red-ox reactions.

Healing Factors

Healing factors comprise compounds that promote or enhance the healingor regenerative process of the tissues on the application site of thecomposition. During the photoillumination of the composition, there isan increase of the absorption of molecules at the treatment site by themucosa. An augmentation in the blood flow at the site of treatment isobserved for an extent period of time. An increase in the lymphaticdrainage and a possible change in the osmotic equilibrium due to thedynamic interaction of the free radical cascades can be enhanced or evenfortified with the inclusion of healing factors. Suitable healingfactors include, but are not limited to:

Hyaluronic acid (Hyaluronan, hyaluronate): is a non-sulfatedglycosaminoglycan, distributed widely throughout connective, epithelialand neural tissues. It is one of the primary components of theextracellular matrix, and contributes significantly to cellproliferation and migration. Hyaluronan is a major component of theskin, where it is involved in tissue repair. While it is abundant inextracellular matrices, it contributes to tissues hydrodynamics,movement and proliferation of cells and participates in a wide number ofcell surface receptor interactions, notably those including primaryreceptor CD44. The hyaluronidases enzymes degrade hyaluronan. There areat least seven types of hyaluronidase-like enzymes in humans, several ofwhich are tumor suppressors. The degradation products of hyaluronicacid, the oligosaccharides and the very-low molecular weight hyaluronicacid, exhibit pro-angiogenic properties. In addition, recent studiesshow that hyaluronan fragments, but not the native high molecular massof hyaluronan, can induce inflammatory responses in macrophages anddendritic cells in tissue injury. Hyaluronic acid is well suited tobiological applications targeting the skin. Due to its highbiocompatibility, it is used to stimulate tissue regeneration. Currentstudies evidenced hyaluronic acid appearing in the early stages ofhealing to physically create room for white blood cells that mediate theimmune response. It is used in the synthesis of biological scaffolds forwound healing applications and in wrinkle treatment. In embodiment, thecomposition includes in the range of less than about 2% hyaluconic acid,or from about 0.001% to about 2%, or preferable from about 0.002% toabout 2%, or from about 0.002% to about 1% hyaluronic acid.

Glucosamine: is one of the most abundant monosaccharides in humantissues and a precursor in the biological synthesis of glycosilatedproteins and lipids. It is commonly used in the treatment ofosteoarthritis. The common form of glucosamine used is its sulfate salt.Glucosamine shows a number of effects including an anti-inflammatoryactivity, stimulation of the synthesis of proteoglycans and thesynthesis of proteolytic enzymes. A suitable range of concentration overwhich glucosamine can be used in the present composition is from lessthan about 5%, or from about 0.0001% to about 5%, or from about 0.0001%to about 3%, and preferable from about 0.001% to about 3%, or from about0.011% to about 1% and about about 1% to about 3%.

Allantoin: is a diureide of glyosilic acid. It has keratolytic effect,increases the water content of the extracellular matrix, enhances thedesquamation of the upper layers of dead (apoptotic) skin cells, andpromotes skin proliferation and wound healing. In embodiment, thecomposition includes in the range of less than about 1% allantoin, orfrom about 0.001% to about 1%, or from about 0.002% to about 1%, orpreferably from about 0.02% to about 1%, and most preferably from about0.02% to about 0.5%.

Also, saffron can act as both a photon-transfer agent and a healingfactor.

Buffering Agents

Buffering agents can be included to promote smear layer removal inclosed pockets and difficult to reach lesions. In certain embodiments,compounds of the present disclosure comprise buffering agents that actas a metal ion quencher and as a buffer. Suitable chelating agents forthe compositions, methods and uses of the disclosure include, but arenot limited to:

Ethylenediaminotetraacetic Acid (EDTA)

Ethylenediaminotetraacetic acid (EDTA) is an amino acid and is used tosequester di- and trivalent metal ions. EDTA binds to metals via fourcarboxylate and two amine groups. EDTA forms especially strong complexeswith Mn(III), Fe(III), Cu(III), Co(III). It is used to buffer solutions.

Ethylene Glycol Tetraacetic Acid (EGTA)

Ethylene glycol tetraacetic acid (EGTA) is related to EDTA, but with amuch higher affinity for calcium than magnesium ions. It is useful formaking buffer solutions that resemble the environment inside livingcells.

Carbonate and Bicarbonate Salts

According to some embodiments, the compositions of the presentdisclosure may optionally further comprise one or more carbonate orbicarbonate salts.

Suitable carbonate or bicarbonate salts that may be present in thecomposition include, but are not limited to: ammonium bicarbonate,caesium bicarbonate, potassium bicarbonate, sodium bicarbonate, cholinebicarbonate, aminoguanidine bicarbonate, tetraethylammonium bicarbonate,barium carbonate, beryllium carbonate, caesium carbonate, calciumcarbonate, cobalt (II) carbonate, copper (II) carbonate, lithiumcarbonate, magnesium carbonate, nickel (II) carbonate, potassiumcarbonate, sodium carbonate, or zinc carbonate.

In some embodiments, the composition of the disclosure comprises one ormore salts selected from bicarbonate salts, carbonate salts or acombination of the foregoing salts. In some embodiments, the compositionof the disclosure comprises one or more bicarbonate salts. In someembodiments when the composition comprises one or more bicarbonatesalts, the bicarbonate salt is sodium bicarbonate. In some embodimentswhen the composition comprises one or more bicarbonate salts, thebicarbonate salt is potassium bicarbonate. In some embodiments, thecomposition of the disclosure comprises one or more carbonate salts. Insome embodiments when the composition comprises one or more carbonatesalts, the carbonate salt is sodium carbonate. In some embodiments whenthe composition comprises one or more carbonate salts, the carbonatesalt is potassium carbonate. In some embodiments when the compositioncomprises one or more carbonate salts, the carbonate salt is calciumcarbonate.

Gelling Agents

Gelling agents for the compositions, uses or methods according to thepresent disclosure may comprise any ingredient suitable for use incomposition as described herein. The gelling agent may be an agentcapable of forming a cross-linked matrix, including physical and/orchemical cross-links. The gelling agent can be biocompatible, and may bebiodegradable. In some embodiments, the gelling agent is able to form ahydrogel or a hydrocolloid. An appropriate gelling agent is one that canform a viscous liquid or a semisolid. In some embodiments, the gellingagent and/or the composition has an appropriate light transmissionproperties. It is also important to select a gelling agent which willallow biophotonic activity of the chromophore(s). For example, somechromophores require a hydrated environment in order to fluoresce. Thegelling agent may be able to form a gel by itself or in combination withother ingredients such as water or another gelling agent, or whenapplied to a treatment site, or when illuminated with light.

The gelling agent according to various embodiments of the presentdisclosure may include, but not be limited to, polyalkylene oxides,particularly polyethylene glycol and poly(ethylene oxide)-poly(propyleneoxide) copolymers, including block and random copolymers; polyols suchas glycerol, polyglycerol (particularly highly branched polyglycerol),propylene glycol and trimethylene glycol substituted with one or morepolyalkylene oxides, e.g., mono-, di- and tri-polyoxyethylated glycerol,mono- and di-polyoxy-ethylated propylene glycol, and mono- anddi-polyoxyethylated trimethylene glycol; polyoxyethylated sorbitol,polyoxyethylated glucose; acrylic acid polymers and analogs andcopolymers thereof, such as polyacrylic acid per se, polymethacrylicacid, poly(hydroxyethylmethacrylate), poly(hydroxyethylacrylate),poly(methylalkylsulfoxide methacrylate), poly(methylalkylsulfoxideacrylate) and copolymers of any of the foregoing, and/or with additionalacrylate species such as aminoethyl acrylate and mono-2-(acryloxy)-ethylsuccinate; polymaleic acid; poly(acrylamides) such as polyacrylamide perse, poly(methacrylamide), poly(dimethylacrylamide), andpoly(N-isopropyl-acrylamide); poly(olefinic alcohol)s such as poly(vinylalcohol); poly(N-vinyl lactams) such as poly(vinyl pyrrolidone),poly(N-vinyl caprolactam), and copolymers thereof, polyoxazolines,including poly(methyloxazoline) and poly(ethyloxazoline); silicones,polyvinyl silicates, tetramethoxyorthosilicates,methyltrimethoxyorthosilicates, tetraalkoxyorthosilicates,trialkoxyorthosilicates, pressure sensitive silicone adhesives (such asBioPSA from Dow-Corning), and polyvinylamines.

The gelling agent according to some embodiments of the presentdisclosure may include a polymer selected from any of synthetic orsemi-synthetic polymeric materials, polyacrylate copolymers, cellulosederivatives and polymethyl vinyl ether/maleic anhydride copolymers. Insome embodiments, the hydrophilic polymer comprises a polymer that is ahigh molecular weight (i.e., molar masses of more than about 5,000, andin some instances, more than about 10,000, or about 100,000, or about1,000,000) and/or cross-linked polyacrylic acid polymer.

In some embodiments, the gelling agent comprises a carbomer. Carbomersare synthetic high molecular weight polymer of acrylic acid that arecross-linked with either allylsucrose or allylethers of pentaerythritolhaving a molecular weight of about 3×10⁶. The gelation mechanism dependson neutralization of the carboxylic acid moiety to form a soluble salt.The polymer is hydrophilic and produces sparkling clear gels whenneutralized. Carbomer gels possess good thermal stability in that gelviscosity and yield value are essentially unaffected by temperature. Asa topical product, carbomer gels possess optimum rheological properties.The inherent pseudoplastic flow permits immediate recovery of viscositywhen shear is terminated and the high yield value and quick break makeit ideal for dispensing. Aqueous solution of Carbopol® is acidic innature due to the presence of free carboxylic acid residues.Neutralization of this solution cross-links and gelatinizes the polymerto form a viscous integral structure of desired viscosity.

Carbomers are available as fine white powders which disperse in water toform acidic colloidal suspensions (a 1% dispersion has a pH ofapproximately 3) of low viscosity. Neutralization of these suspensionsusing a base, for example sodium, potassium or ammonium hydroxides, lowmolecular weight amines and alkanolamines, results in the formation oftranslucent gels. Nicotine salts such as nicotine chloride form stablewater-soluble complexes with carbomers at about pH 3.5 and arestabilized at an optimal pH of about 5.6.

In some embodiments of the disclosure, the carbomer is Carbopol®. Suchpolymers are commercially available from B.F. Goodrich or Lubrizol underthe designation Carbopol® 71G NF, 420, 430, 475, 488, 493, 910, 934,934P, 940, 971PNF, 974P NF, 980 NF, 981 NF and the like. Carbopols areversatile controlled-release polymers, as described by Brock(Pharmacotherapy, 14:430-7 (1994), incorporated herein by reference) andDurrani (Pharmaceutical Res. (Supp.) 8:S-135 (1991), incorporated hereinby reference), and belong to a family of carbomers which are synthetic,high molecular weight, non-linear polymers of acrylic acid, crosslinkedwith polyalkenyl polyether. In some embodiments, the carbomer isCarbopol® 974P NF, 980 NF, 5984 EP, ETD 2020NF, Ultrez 10 NF, 934 NF,934P NF or 940 NF. In some embodiments, the carbomer is Carbopol® 980NF, ETD 2020 NF, Ultrez 10 NF, Ultrez 21 or 1382 Polymer, 1342 NF, 940NF. In some embodiments, about 0.05% to about 10%, about 0.5% to about5%, or about 1% to about 3% by weight of the final composition of a highmolecular weight carbopol can be present as the gelling agent. In someembodiments, the composition of the disclosure comprises about 0.05% toabout 10%, about 0.5% to about 5%, or about 1% to about 3% by weight ofthe final composition of a high molecular weight carbopol.

In some embodiments, the gelling agent comprises a hygroscopic and/or ahydrophilic material useful for their water attracting properties. Thehygroscopic or hydrophilic material may include, but is not limited to,glucosamine, glucosamine sulfate, polysaccharides, cellulose derivatives(hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxypropylcellulose, methylcellulose and the like), noncellulose polysaccharides(galactomannans, guar gum, carob gum, gum arabic, sterculia gum, agar,alginates and the like), glycosaminoglycan, poly(vinyl alcohol),poly(2-hydroxyethylmethylacrylate), polyethylene oxide, collagen,chitosan, alginate, a poly(acrylonitrile)-based hydrogel, poly(ethyleneglycol)/poly(acrylic acid) interpenetrating polymer network hydrogel,polyethylene oxide-polybutylene terephthalate, hyaluronic acid,high-molecular-weight polyacrylic acid, poly(hydroxy ethylmethacrylate),poly(ethylene glycol), tetraethylene glycol diacrylate, polyethyleneglycol methacrylate, and poly(methyl acrylate-co-hydroxyethyl acrylate).In some embodiments, the hydrophilic gelling agent is selected fromglucose, modified starch, methyl cellulose, carboxymethyl cellulose,propyl cellulose, hydroxypropyl cellulose, carbomers, alginic acid,sodium alginate, potassium alginate, ammonium alginate, calciumalginate, agar, carrageenan, locust bean gum, pectin, and gelatin.

The gelling agent may be protein-based/naturally derived material suchas sodium hyaluronate, gelatin or collagen, lipids, or the like. Thegelling agent may be a polysaccharide such as starch, chitosan, chitin,agarose, agar, locust bean gum, carrageenan, gellan gum, pectin,alginate, xanthan, guar gum, and the like.

In some embodiments, the composition can include up to about 2% byweight of the final composition of sodium hyaluronate as the singlegelling agent. In some embodiments, the composition can include morethan about 4% or more than about 5% by weight of the final compositionof gelatin as the single gelling agent. In some embodiments, thecomposition can include up to about 10% or up to about 8% starch as thesingle gelling agent. In some embodiments, the composition can includemore than about 5% or more than about 10% by weight of the finalcomposition of collagen as the gelling agent. In some embodiments, about0.1% to about 10% or about 0.5% to about 3% by weight of the finalcomposition of chitin can be used as the gelling agent. In someembodiments, from about 0.5% to about 5% by weight of the finalcomposition of corn starch or from about 5% to about 10% by weight ofthe final composition of corn starch can be used as the gelling agent.In some embodiments, more than about 2.5 wt % by weight of the finalcomposition of alginate can be used in the composition as the gellingagent. In some embodiments, the percentages by weight percent of thefinal composition of the gelling agents can be as follows: cellulose gel(from about 0.3% to about 2.0%), konjac gum (from about 0.5% to about0.7%), carrageenan gum (from about 0.02% to about 2.0%), xanthan gum(from about 0.01% to about 2.0%), acacia gum (from about 3% to about30%), agar (from about 0.04% to about 1.2%), guar gum (from about 0.1%to about 1%), locust bean gum (from about 0.15% to about 0.75%), pectin(from about 0.1% to about 0.6%), tara gum (from about 0.1% to about1.0%), polyvinylypyrrolidone (from about 1% to about 5%), sodiumpolyacrylate (from about 1% to about 10%). Other gelling agents can beused in amounts sufficient to gel the composition or to sufficientlythicken the composition. It will be appreciated that lower amounts ofthe above gelling agents may be used in the presence of another gellingagent or a thickener.

The composition of the present disclosure may be further encapsulated,e.g., in a membrane. Such a membrane may be transparent, and/orsubstantially, or fully impermeable. The membrane may be impermeable toliquid but permeable to gases such as air. In some embodiments, thecomposition may form a membrane that encapsulates the chromophore(s) ofthe biophotonic topical composition, where the membrane may besubstantially impermeable to liquid and/or gas. The membrane may beformed of one or more lipidic agents, polymers, gelatin, cellulose orcyclodextrins, or the like. In some embodiments, the membrane istranslucent or transparent to allow light to infiltrate to and from thechromophore(s). In some embodiments, the composition is a dendrimer withan outer membrane comprising poly(propylene amine). In some embodiments,the outer membrane comprises gelatin.

Polyols

According to some embodiments, the compositions of the methods and usesof the present disclosure may optionally further comprise one or morepolyols. Suitable polyols that may be included in the compositioninclude, but are not limited to a diol, a triol, a saccharide,glycerine, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol,propylene glycol, butanediol, butenediol, butynediol, pentanediol,hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol,diethylene glycol, triethylene glycol, tetraethylene glycol, dipropyleneglycol and dibutylene glycol. In some embodiments when the compositionof the disclosure includes one or more polyols, the polyol is glycerine.In some embodiments when the composition of the disclosure includes oneor more polyols, the polyol is propylene glycol. In some embodimentswhen the composition of the disclosure includes one or more polyols, thepolyol is a combination of glycerine and propylene glycol.

In some embodiments, one or more polyols are present in an amount ofabout 5-75% by weight of the total composition, such as 5-75% by weightof the total composition. In some embodiments, one or more polyols arepresent in an amount of about 10-75% by weight of the total composition,such as 10-75% by weight of the total composition. In some embodiments,one or more polyols are present in an amount of about 15-75% by weightof the total composition, such as 15-75% by weight of the totalcomposition. In some embodiments, one or more polyols are present in anamount of about 20-75% by weight of the total composition, such as20-75% by weight of the total composition.

Additional Components

The compositions, methods, and uses of the disclosure can also includeother ingredients such as humectants (e.g., glycerine, ethylene glycol,and propylene glycol), preservatives such as parabens, and pH adjusterssuch as sodium hydroxide, sodium bicarbonate, and HCl. In someembodiments, the pH of the composition is in or adjusted to the range ofabout 4 to about 10. In some embodiments, the pH of the composition isin or adjusted to the range of about 4 to about 9. In some embodiments,the pH of the composition is in or adjusted to the range of about 4 toabout 8. In some embodiments, the pH of the composition is within therange of about 4 to about 7. In some embodiments, the pH of thecomposition is within the range of about 4 to about 6.5. In someembodiments, the pH of the composition is within the range of about 4 toabout 6. In some embodiments, the pH of the composition is within therange of about 4 to about 5.5. In some embodiments, the pH of thecomposition is within the range of about 4 to about 5. In someembodiments, the pH of the composition is within the range of about 5.0to about 8.0. In some embodiments, the pH of the composition is withinthe range of about 6.0 to about 8.0. In some embodiments, the pH of thecomposition is within the range of about 6.5 to about 7.5. In someembodiments, the pH of the composition is within the range of about 5.5to about 7.5.

In some embodiments, the pH of the composition is in or adjusted to therange of 4 to 10. In some embodiments, the pH of the composition is inor adjusted to the range of 4 to 9. In some embodiments, the pH of thecomposition is in or adjusted to the range of 4 to 8. In someembodiments, the pH of the composition is within the range of 4 to 7. Insome embodiments, the pH of the composition is within the range of 4 to6.5. In some embodiments, the pH of the composition is within the rangeof 4 to 6. In some embodiments, the pH of the composition is within therange of 4 to 5.5. In some embodiments, the pH of the composition iswithin the range of 4 to 5. In some embodiments, the pH of thecomposition is within the range of 5.0 to 8.0. In some embodiments, thepH of the composition is within the range of 6.0 to 8.0. In someembodiments, the pH of the composition is within the range of 6.5 to7.5. In some embodiments, the pH of the composition is within the rangeof 5.5 to 7.5.

In some embodiments, the compositions of the disclosure also include anaqueous substance (water) or an alcohol. Alcohols include, but are notlimited to, ethanol, propanol, isopropanol, butanol, iso-butanol,t-butanol or pentanol. In some embodiments, the chromophore orcombination of chromophores is in solution in a medium of thecomposition. In some embodiments, the chromophore or combination ofchromophores is in solution in a medium of the composition, wherein themedium is an aqueous substance.

Methods of Use and Treatment

As discussed above, biofilms are mucilaginous communities ofmicroorganisms such as bacteria, archaea, fungi, molds, algae orprotozoa or mixtures thereof that grow on various surfaces. Biofilmsform when microorganisms establish themselves on a surface and activategenes involved in producing a matrix that includes polysaccharides. Thismatrix may provide protection of biofilm forming bacteria from biocides.

Microbial cells can grow as a single-cell free living organism(planktonic) or as cell aggregates in the form of biofilms. Biofilms arephysiologically distinct from planktonic cells of the same organism,which, by contrast, are single-cells that may float or swim in a liquidmedium. Biofilms are composed of highly organized communities ofmicroorganisms which are surrounded by an extracellular matrix (ECM).The ECM is typically a microbial-derived complex comprisingpolysaccharides, proteins, lipids, and nucleic acids. Additionally, theECM may contain host-derived components such as fibrin, platelets, andimmunoglobulins (see, Harper, D. R. et. al; “Bacteriophages andBiofilms”, Antibiotics, 2015, 3, 270-284;doi:10.3390/antibiotics3030270)

Biofilms can be formed by different microorganism species and generaincluding bacteria, archaea, fungi, molds, algae or protozoa or mixturesthereof that grow on various surfaces. Bacteria involved in theformation of biofilms include both gram negative and gram positivebacteria. Gram-negative bacteria are a group of bacteria which do notabsorb crystal violet during the Gram stain test and include bacteriasuch as Pseudomona aeruginosa (P. aeruginosa), Escherichia coli (E.coli), Neisseria gonorrhoeae, Chlamydia trachomatis, Proteus mirabilis,and Yersinia pestis. Gram-positive bacteria are a group of bacteriawhich absorb crystal violet during the Gram stain test. TheGram-positive bacteria typically have thick peptidoglycan layer in thebacterial cell wall retains the stain. Some exemplary examples ofGram-positive bacteria include Staphylococcus aureus (S. aureus),Bacillus spp, Listeria monocytogenes, Staphylococcus spp, and lacticacid bacteria, including Lactobacillus plantarum and Lactococcus lactis.

Biofilm-related infections can be divided into two major groups:device-related or tissue-related. Device-related biofilms are involvedin many hospital-acquired infections in which they are known tocontaminate devices such as implants and catheters and to preventadequate antimicrobial treatment of such devices. Tissue-relatedbiofilms often result in adverse health conditions following theirinvolvement in diseases such as chronic obstructive pulmonary disease,tuberculosis, chronic wound infections, chronic otitis media, chronicsinusitis, lung infections such as cystic fibrosis, and tooth decay.

The duration of the exposure to actinic light will be dependent on thesurface of the treated area, and on the type of lesion, trauma or injurythat is being treated. The illumination of the composition may takeplace within seconds or even fragment of seconds, but a prolongedexposure period is beneficial to exploit the synergistic effects of theabsorbed, reflected and reemitted light on the composition of thepresent disclosure and its penetration in the tissue being treated. Insome embodiments, the time of exposure to actinic light of the tissue onwhich the composition has been applied is a period of less than 5minutes. In other embodiments, the time of exposure is from about 20seconds to about 5 minutes, or from between about 60 second and about 5minutes. In other embodiments, the time of exposure to actinic light ofthe tissue on which the composition has been applied is a period of lessthan about 5 minutes. In other embodiments, the time of exposure isbetween about 20 seconds to about 5 minutes, or between about 60 secondsand about 5 minutes per cm² of the area to be treated, so that the totaltime of exposure of a 10 cm² area would be between 10 minutes and 50minutes. In yet other embodiments, the source of actinic light is incontinuous motion over the treated area for the appropriate time ofexposure. In yet other embodiments, multiple applications of thecomposition and actinic light are performed. In some embodiments, thetissue, skin or wound is exposed to actinic light at least two, three,four, five or six times, or more, depending on the patient'srequirement. Also, the entire treatment may be repeated in its entiretyas may be required by the patient. In some embodiments, a freshapplication of the composition is applied before exposure to actiniclight.

Combination Treatments

In some embodiments, compositions of the present disclosure may be usedwith other therapeutic treatments. The phrase “combination therapy”embraces the application of any of the compositions described herein,and an additional therapeutic regime, or combination of them, as part ofa specific treatment regimen intended to provide a beneficial effectfrom the co-action of these therapeutic regime. Application of thesetherapeutic regimes in combination typically is carried out over adefined time period (usually minutes, hours, days or weeks dependingupon the combination selected). Typically, “combination therapy” isintended to embrace application of these therapeutic regimes in asequential manner, that is, wherein each regimen is applied at adifferent time, as well as, or alternatively application of theseregimes, or at least two of the compositions, in a substantiallysimultaneous manner. The therapeutic agents can be applied by the sameroute or by different routes. Alternatively, for example, alltherapeutic agents may be applied topically. “Combination therapy” alsocan embrace the application of the compositions as described herein infurther combination with other biologically active ingredients (such as,but not limited to, a second and different therapeutic agent) andnon-drug therapies (such as, but not limited to, surgery or radiation).

In some embodiments, the therapeutic agents utilized in a combinationtherapy may be utilized simultaneously, separately, or sequentially withany of the compounds and compositions of this disclosure, or mixturesthereof, and may comprise, but are not limited to: a non-steroidalanti-inflammatory drug (NSAID), an anti-inflammatory agent, acorticosteroid, an anti-allergic agent, a steroid drug, one or more ofthe antimicrobial agents described above, one or more collagens and/oragents that promote collagen synthesis described above, or mixturesthereof.

In some embodiments, any of the compositions described herein can allowthe combination therapeutic agents and/or compositions described hereinor mixtures thereof to be utilized at a low dose, that is, at a doselower than has been conventionally used in clinical situations.

Alternatively, the methods and combinations of this disclosure may allowfor maximizing a therapeutic effect at higher doses.

In some embodiments, when utilized as a combination, the therapeuticagents can be formulated as separate compositions which are given at thesame time or different times, or the therapeutic agents can be given asa single composition.

Kits

The present disclosure also provides kits for preparing and/or applyingany of the compositions of the present disclosure for inhibiting biofilmformation or disrupting existing or developing biofilms in a subject.The kit may include a composition as described above (e.g., abiophotonic topical composition), and may also include a light source,an apparatus for applying or removing the composition, and instructionsof use for the composition and/or a light source. In some embodiments,the composition comprises at least one oxidant and at least onechromophore capable of activating the oxidant. In other embodiments, thecomposition comprises at least one chromophore and at least one saltselected from bicarbonate salts, carbonate salts, or a combination offoregoing salts.

In some embodiments, the kit includes more than one composition, forexample, a first and a second composition. The first composition mayinclude at least one chromophore and the second composition may includeat least one oxidant. In certain such embodiments, the oxidant is chosenfrom hydrogen peroxide, carbamide peroxide and benzoyl peroxide. Incertain such embodiments, the first and/or second composition furthercomprises one or more gelling agents.

In some embodiments, the kit includes more than one composition, forexample, a first and a second composition. The first composition mayinclude at least one chromophore and the second composition may includeat least one salt selected from bicarbonate salts, carbonate salts, or acombination of foregoing salts. In certain such embodiments, the firstand/or second composition further comprises one or more gelling agents.

In some embodiments, the first composition may comprise the at least onechromophore in a liquid or as a powder, and the second composition maycomprise at least one oxidant. In certain such embodiments, the oxidantis chosen from hydrogen peroxide, carbamide peroxide and benzoylperoxide. In certain such embodiments, the first and/or secondcomposition further comprises one or more gelling agents.

In some embodiments, the first composition may comprise the at least onechromophore, and the second composition may comprise at least one saltselected from bicarbonate salts, carbonate salts, or a combination offoregoing salts. In certain such embodiments, the first and/or secondcomposition further comprises one or more gelling agents.

In some embodiments, the kit includes containers comprising thecompositions of the present disclosure. In some embodiments, the kitincludes a first container comprising the at least one chromophore and asecond container comprising at least one oxidant. In certain suchembodiments, the oxidant is chosen from hydrogen peroxide, carbamideperoxide and benzoyl peroxide. In certain such embodiments, the firstand/or second composition further comprises one or more gelling agents.

In some embodiments, the kit includes containers comprising thecompositions of the present disclosure. In some embodiments, the kitincludes a first container comprising the at least one chromophore and asecond container comprising at least one salt selected from bicarbonatesalts, carbonate salts, or a combination of foregoing salts. In certainsuch embodiments, the first and/or second composition further comprisesone or more gelling agents.

The containers may be light impermeable, air-tight and/or leakresistant. Exemplary containers include, but are not limited to,syringes, vials, or pouches. The first and second compositions may beincluded within the same container but separated from one another untila user mixes the compositions. In some embodiments, the container may bea dual-chamber syringe where the contents of the chambers mix onexpulsion of the compositions from the chambers. In some embodiments,the pouch may include two chambers separated by a frangible membrane. Insome embodiments, one component may be contained in a syringe andinjectable into a container comprising the second component.

The composition may also be provided in a container comprising one ormore chambers for holding one or more components of the composition, andan outlet in communication with the one or more chambers for dischargingthe composition from the container.

In some embodiments, the kit comprises a systemic or topical drug foraugmenting the treatment of the composition. For example, in certainsuch embodiments, the kit may include a systemic or topical agent, e.g.,an anesthetics or anti-inflammation agent, for reducing pain.

Written instructions on how to use the composition in accordance withthe present disclosure may be included in the kit, or may be included onor associated with the containers comprising the compositions of thepresent disclosure.

In some embodiments, the kit may comprise a further component which is adressing. The dressing may be a porous or semi-porous structure forreceiving the composition. The dressing may comprise woven or non-wovenfibrous materials.

In some embodiments of the kit, the kit may further comprise a lightsource such as a portable light with a wavelength appropriate toactivate the chromophore in the composition. The portable light may bebattery operated or re-chargeable.

In some embodiments, the kit may further comprise one or morewaveguides.

Identification of equivalent compositions, methods and kits are wellwithin the skill of the ordinary practitioner and would require no morethan routine experimentation, in light of the teachings of the presentdisclosure. Practice of the disclosure will be still more fullyunderstood from the following examples, which are presented herein forillustration only and should not be construed as limiting the disclosurein any way.

EXAMPLES

The examples below are given so as to illustrate the practice of variousembodiments of the present disclosure. They are not intended to limit ordefine the entire scope of this disclosure.

It should be appreciated that the disclosure is not limited to theparticular embodiments described and illustrated herein but includes allmodifications and variations falling within the scope of the disclosureas defined in the appended embodiments.

Example 1—Biofilm Formation Elimination/Reduction & Inhibition ofBacterial Biofilm Species Found in Wounds

A study was performed to evaluate an anti-biofilm activity of abiophotonic composition of the present disclosure on a biofilm generatedby a Gram-negative, rod-shaped bacterium, e.g., Pseudomonas aeruginosaor Proteus mirabilis, and a biofilm produced by a Gram-positive coccalbacterium, e.g., Staphylococcus aureus. Each of these bacterial speciesis a major cause of wound infection and wound-associated biofilmformation.

Specifically, experiments were performed to assess an effect ofillumination on the effectiveness of the biophotonic composition toreduce the presence of a wound biofilm and also the presence of thewound-associated bacterial species (see, DeLeon et al. (2014)“Synergistic Interactions of Pseudomonas aeruginosa and Staphylococcusaureus in an In Vitro Wound Model”, Infection and Immunity, 82(11):4718-4728). In addition, experiments were performed to assess the effectof varying an amount of an oxidant (e.g., urea peroxide (UP), alsoreferred to as carbamide peroxide) in the biophotonic composition andthe effect of such a variation on the ability of the biophotoniccomposition to reduce the presence of a wound biofilm and the presenceof the wound-associated bacterial species. Illumination of thebiophotonic composition was carried out with a multi-LED lamp (THERA™lamp) emitting non-coherent blue light in a wavelength range of 400 to470 nm with the lamp placed at a distance of 5 cm from the givenbiofilm.

Preparation of Bacterial Biofilms

A biofilm of the given bacterial species was prepared in accordance withthe methodology published in Javanbakht et al. (2016) “Relating theSurface Properties of Supraparamegnetic Iron Oxide Nanoparticles(SPIONS) to Their Bactericidal Effect towards a Biofilm of Streptococcusmutans” PLOS ONE, 11(4), e0154445. Doi:10.1371/journal.pone.0154445.

Day 1: Bacteria (P. aeruginosa, P. mirabilis, or S. aureus) wereinoculated on TYE medium agar plates (diameter of plates: 10 cm) and theplates were incubated overnight in dark at 37° C. to allow for coloniesto grow.

Day 2: A sample was prepared containing 5 mL of TYE liquid culturalmedium, 25 μL of glucose (40% concentration). A small quantity ofbacteria from the plate were inoculated into the liquid media. Theliquid sample was incubated overnight at 37° C. overnight in dark.

Day 3: An aliquot of the liquid medium containing overnight culture fromday 2 was withdrawn and an absorbance reading was taken of the aliquotto determine whether sufficient bacterial cell growth had occurred inorder to proceed. Thereafter, a diluted sample was prepared containing8.875 mL of cultural medium, 125 μL of saccharose (40%) and 1 mL of thebacterial sample prepared in day 2. 500 μL of the diluted solution ofbacteria was distributed in each well of a FCS plate and the plate wasleft to incubate in the oven at 37° C. overnight.

Day 4: Confocal microscopy was performed on the given bacterial biofilmusing a Leica TCS SP5 confocal laser scanning microscope.

Bacterial Cell Counting

The absorbance value of the bacterial cells was determined by a HACH DR2800 spectrometer (0.84 AU). The concentration of bacterial cells in theliquid TYE culture medium containing 0.2% glucose and incubated for 24 hin the dark at 37° C. was 1.0×10⁷ bacteria per mL (see, Javanbakht etal. (2016)).

Quantification of the Bacteria in the Biofilms

To assess whether a biophotonic composition of the present disclosurecould have an anti-biofilm effect, a 120 mg sample of the givencomposition was placed into the biofilm bearing well on the FCS plate.Prior to adding the composition to the well, the bacteria in the wellwere quantified. The measured thickness of the biofilms of P.aeruginosa, P. mirabilis, and S. aureus were 7.0 μm and 14.0 μm,respectively. For P. mirabilis only, the BacLight stains were addedafter the treatment with the biophotonic composition. These biofilmthicknesses are in accordance with biofilms produced by each of thesebacterial species as found, for example, in Dong et al. (2015)“Distribution and Inhibition of Liposomes on Staphylococcus aureus andPseudomonas aeruginosa Biofilm” PLoS ONE 10(6): e0131806.Doi10.1371/journal-pone.0131806. The thickness of the added gels was 1.5mm.

A BacLight kit (Invitrogen) technique was used to evaluate, bymicroscopic in situ image analysis, the presence of live and deadbacteria to assess each of the tested compositions' anti-biofilm effect.The methodology utilized was in accordance with that described inJavanbakht et al. (2016), with the live and dead bacteria being stainedgreen and red, respectively, whereas a third image in a set was a mergedimage of the live and dead cells. Two types of controls were used: thefirst contained the bacterial cells alone and the second controlcontained the bacterial cells incubated with the BacLight kit.

Biological Assessment

A confocal fluorescence microscope (Leica TCS SP5) was used for in situfor the biofilms evaluations. As noted above, cell viability wasdetermined with a standard Live/Dead BacLight (Invitrogen) test usingSYTO9-propidium iodide. Cell numbers were quantified using a HACH DR2800 spectrometer using DR2700 software. Biofilms were stained withpropidium iodide and Syto 9 for 15 minutes, prior to observation [see,Javanbakht et al. (2016), and also Zhang et al. (2011), “QuantifyingDiffusion in a Biofilm of Streptococcus mutans”, Antimicrobial Agentsand Chemotherapy, 55(3): 1075-1081)] before adding 120 mg of thecomposition. The experiments for P. aeruginosa, P. mirabilis, or S.aureus were carried out in triplicate and the imaging of samples wascarried out fifteen times and the standard deviations of the ratio ofdead to total cells were calculated.

Results and Discussion

Quantification of the Bacteria in the Biofilms

Tables 1-4 show the percentages of bacterial mortality in the biofilmsof P. aeruginosa, P. mirabilis, or S. aureus as a function of thepresence or absence of chromophore, the presence or absence of UP (withvarying %), and illumination with the THERA™ lamp

TABLE 1 Percentages of bacterial mortality of Pseudomonas aeruginosa.Increase of Concentration bacterial Experimental of Duration ofmortality condition UP (%) in gel illumination (%) Gel lacking 0 Noillumination 12.3 ± 2.1 chromophore Gel lacking 0 No illumination 14.1 ±1.7 chromophore + carbopol gel Gel lacking 0 5 minutes 16.4 ± 4.5chromophore + carbopol gel Gel with chromophore 0 No illumination 21.4 ±3.4 (Eosin Y at 0.305 mg/ml) + carbopol gel Gel with chromophore 0 5minutes 31.9 ± 4.3 (Eosin Y at 0.305 mg/ml) + carbopol gel Gel withchromophore 1 No illumination 24.6 ± 1.5 (Eosin Y at 0.305 mg/ml) +carbopol gel with UP Gel with chromophore 1 5 minutes 42.5 ± 2.3 (EosinY at 0.305 mg/ml) + carbopol gel with UP Gel with chromophore 3 Noillumination 31.2 ± 1.9 (Eosin Y at 0.305 mg/ml) + carbopol gel with UPGel with chromophore 3 5 minutes 48.4 ± 1.5 (Eosin Y at 0.305 mg/ml) +carbopol gel with UP Gel with chromophore 6 No illumination 35.9 ± 2.4(Eosin Y at 0.305 mg/ml) + carbopol gel with UP Gel with chromophore 6 5minutes 50.5 ± 1.8 (Eosin Y at 0.305 mg/ml) + carbopol gel with UP Gelwith chromophore 12 No illumination 49.5 ± 3.5 (Eosin Y at 0.305mg/ml) + carbopol gel with UP Gel with chromophore 12 5 minutes 53.7 ±2.6 (Eosin Y at 0.305 mg/ml) + carbopol gel with UP

TABLE 2 Percentages of bacterial mortality of Staphylococcus aureus.Increase of Concentration Duration bacterial Experimental of of photo-mortality condition UP (%) induction (%) Gel lacking 0 No illumination 9.5 ± 2.8 chromophore Gel lacking 0 No illumination 16.1 ± 2.0chromophore + carbopol gel Gel lacking 0 5 minutes 20.9 ± 3.8chromophore + carbopol gel Gel with chromophore 0 No illumination 22.4 ±2.8 (Eosin Y at 0.305 mg/ml) + carbopol gel Gel with chromophore 0 5minutes 34.2 ± 2.9 (Eosin Y at 0.305 mg/ml) + carbopol gel Gel withchromophore 1 No illumination 30.8 ± 1.3 (Eosin Y at 0.305 mg/ml) +carbopol gel with UP Gel with chromophore 1 5 minutes 33.6 ± 2.4 (EosinY at 0.305 mg/ml) + carbopol gel with UP Gel with chromophore 3 Noillumination 32.3 ± 1.7 (Eosin Y at 0.305 mg/ml) + carbopol gel with UPGel with chromophore 3 5 minutes 35.5 ± 3.0 (Eosin Y at 0.305 mg/ml) +carbopol gel with UP Gel with chromophore 6 No illumination 34.9 ± 1.8(Eosin Y at 0.305 mg/ml) + carbopol gel with UP Gel with chromophore 6 5minutes 46.9 ± 2.6 (Eosin Y at 0.305 mg/ml) + carbopol gel with UP Gelwith chromophore 12 No illumination 38.4 ± 2.6 (Eosin Y at 0.305mg/ml) + carbopol gel with UP Gel with chromophore 12 5 minutes 55.6 ±2.3 (Eosin Y at 0.305 mg/ml) + carbopol gel with UP

TABLE 3 Percentages of bacterial mortality of Proteus mirabilis AverageGel Thera ™ Lamp % dead SD Control (Set 1) — 8.1 5.1 Chromo + Carrier UP0 1 min 36.6 19.1 Chromo + Carrier UP 1 1 min 55.4 13.5 Chromo + CarrierUP 3 1 min 53.1 25.6 Chromo + Carrier UP 6 1 min 48.3 27.0 Chromo +Carrier UP 12 1 min 49.3 23.6 Control (Set 2) — 8.1 5.1 Chromo + CarrierUP 0 2 min 49.6 18.3 Chromo + Carrier UP 1 2 min 62.1 14.7 Chromo +Carrier UP 3 2 min 59.8 7.0 Chromo + Carrier UP 6 2 min 52.0 16.4Chromo + Carrier UP 12 2 min 53.8 19.0 Control (Set 3) — 22.4 14.9Chromo + Carrier UP 0 5 min 62.1 26.7 Chromo + Carrier UP 1 5 min 92.111.7 Chromo + Carrier UP 3 5 min 88.3 13.7 Chromo + Carrier UP 6 5 min92.9 9.9 Chromo + Carrier UP 12 5 min 94.6 8.2 Control (Set 4) — 21.510.9 Chromo Carrier UP 1 5 min 40.0 22.6 Placebo + Chromo Carrier UP 3 5min 36.1 25.5 Placebo + Chromo Carrier UP 6 5 min 40.3 21.2 Placebo +Chromo Carrier UP 12 5 min 47.0 30.3 Placebo + Control (Set 5) — 10.35.2 Chromo Carrier Placebo 1 9.2 6.3 Placebo+ Chromo Carrier Placebo 318.6 4.9 Placebo+ Chromo Carrier Placebo 5 20.2 21.2 Placebo+ ChromoCarrier Placebo 10  24.4 24.4 Placebo+ Control (Set 6) — 12.0 5.4Chromo + Carrier Placebo 1 22.8 4.6 Chromo + Carrier Placebo 3 31.3 13.9Chromo + Carrier Placebo 5 45.4 43.6 Chromo + Carrier Placebo 10  32.012.7 Control (Set 7) — 12.0 5.4 Chromo + Carrier UP 12 1 68.2 29.5Chromo + Carrier UP 12 3 79.0 10.4 Chromo + Carrier UP 12 5 95.2 1.2Chromo + Carrier UP 12 10  95.7 7.4 SD = Standard Deviation; “Carrier UP12” indicates carrier gel (carbopol gel) with 12% UP concentration(carrier gel bearing a given % of UP are similarly indicated as such);“carrier placebo” indicates carrier without UP; “chromo placebo+”indicates gel with no chromophore.

TABLE 4 Percentages of bacterial mortality for P. mirabilis, P.aeruginosa, and S. aureus Average Gel Thera ™ Lamp % dead SD Control(Set P. mirabilis) — 11.8 11.0 Chromo + Carrier UP 12 — 33.0 30.1Chromo + Carrier UP 12 5 min 92.7 19.8 Chromo + Carrier UP 12 — 28.736.4 Placebo Chromo + Carrier UP 12 5 min 41.3 37.4 Placebo Control (SetP. aeruginosa) — 40.5 19.4 Chromo + Carrier UP 12 — 77.0 13.2 Chromo +Carrier UP 12 5 min 90.1 8.2 Chromo + Carrier UP 12 — 62.1 15.2 PlaceboChromo + Carrier UP 12 5 min 74.9 21.0 Placebo Control (Set S. aureus) —10.2 5.2 Chromo + Carrier UP 12 — 32.5 21.1 Chromo + Carrier UP 12 5 min92.5 4.4 Chromo + Carrier UP 12 — 16.0 11.8 Placebo Chromo + Carrier UP12 5 min 21.3 14.3 Placebo

As shown in Tables 1 and 2, while there was an increase of the bacterialmortality with the illumination of the non-chromophore/no-UPcompositions (compared to the non-illuminated controls), there was asignificantly larger increase in the bacterial cell mortality in thepresence of the chromophore-containing compositions for both bacterialspecies, with the mortality being consistently higher in the illuminatedtest conditions than non-illuminated conditions. Regarding the P.aeruginosa biofilms, the bacterial cell mortality increased when thebiophotonic composition contained an increasing amount of UP with aplateau level of 6% UP (although the mortality was still higher with the12% UP biophotonic gel composition); for the S. aureus biofilms, thebiophotonic gel composition with increasing amounts of UP yieldedsimilar results to those of the P. aeruginosa biofilms, both in terms ofnon-illumination and illuminated test conditions, and with an increasingof the UP concentration with the effect being more pronounced at a 12%UP level. Mortality for P. mirabilis showed similar trends (Table 3).Nevertheless, bacterial mortality for P. aeruginosa, P. mirabilis, andS. aureus was generally higher in the presence of biophotonic gelcompositions and illumination, even in the absence of UP (see, generallyTables 1-3). Moreover, confocal microscopy studies showed thatbiophotonic gel composition combined with 12% UP and illuminated for 5minutes exerted comparable mortality on S. aureus as 70% ethanol (datanot shown). Further, the biophotonic composition with illuminationshowed reduction in bacterial colonization in venous leg ulcers (datanot shown), as determined by MolecuLighti:X™.

As shown in FIG. 1, the effect of treating P. aeruginosa biofilm with abiophotonic composition of the present disclosure, when illuminated, wasgreater in comparison to treating with either the control composition orjust illumination-alone (illumination in the presence of anon-chromophore/no-UP gel). A similar result was observed in theexperiment with S. aureus biofilm, with data shown in FIG. 2.

Example 2—Biofilm Reduction/Elimination—Chronic Wounds Treated with aBiophotonic Gel Composition

The study was designed to recruit a total of 100 patients at 12 clinicalsites, but only 99 patients were included in total in the finalanalysis. As noted herein, reference is sometimes made to an “interimstudy” which includes an initial subset of 33 patients for which datawere initially available. In certain instances, data from the interimstudy are disclosed herein.

An initial screen visit to identify eligible patients was conductedprior to beginning treatment. The recruited patients' would beparticipating for their treatment of chronic wounds (pressure ulcers(PUs), venous leg ulcers (VLUs) and diabetic foot ulcers (DFUs) in areal-life context clinical setting using a biophotonic composition ofthe present disclosure comprising a synthetic chromophore (Eosin Y) andan oxidant in a carbopol-based gel. For this study, the treatment periodwas designed to be two applications of the biophotonic composition perweek over a period of treatment of 16 weeks for both PUs and VLUs, andover a period of 24 weeks for DFUs. A follow-up period of 8-weeks,commencing upon the termination of treatment, was used to confirm thepersistence of wound closure, once a wound has closed.

Patient's ulcer was first cleansed with normal saline. If needed, sharpdebridement was performed, to remove excess necrotic tissue or foreignmaterials. The biophotonic composition was then prepared, and anapproximately 2 mm thick layer of the composition was applied on thesurface of the ulcer. The patient was then supplied with eye protectorsand the persons administering the treatment were requested to weargoggles, to protect their eyes. The KLOX Multi-LED light (THERA™ lamp)delivering non-coherent blue light in a range 400-470 nm was then placedover the wound area, at a measured distance of 5 cm, using the attachedlamp measuring probe. The ulcer was illuminated for a period of 5minutes. Once the illumination period was completed, the composition wasremoved from the wound surface with a sterile spatula. The wound wasthen wiped with a moist towel or gauze, and then irrigated with salinesolution. A non-adherent dressing was applied, either self-fixating orfixed to the patient with tape and/or bandages, to prevent any contactbetween the wound and the external environment. Local standard of carewas then followed.

Efficacy was assessed using the following endpoints:

-   -   Rate of complete wound closure (wound closure being defined as        skin re-epithelialization without drainage or dressing        requirements confirmed at two consecutive visits, two weeks        apart);    -   Rate of complete wound closure by week 16 for PUs and VLUs, and        by week 24 for DFUs;    -   Time to complete wound closure;    -   Wound area reduction over time;    -   Wound volume reduction over time;    -   Incidence of wound breakdown, following closure;    -   Impact of treatment on patients' quality of life;    -   Ease of use by healthcare professionals.

From the one hundred screened patients who enrolled in the study, atotal of ninety-nine were enrolled and treated, at least once. Of thethirty-three patients in the interim study, sixteen patients completedthe study period as per protocol, while seventeen patients who met thecriteria were enrolled and treated, but discontinued early. Among theseventeen patients who early discontinued from the study, eleven werebeing treated for diabetic foot ulcers (DFUs), two were being treatedfor pressure ulcers (PUs) and four were being treated for venous legulcers (VLUs).

With respect to the demographic and other baseline criteria for theninety-nine patients of the trial, the mean age of the patients was68.70 years, with the youngest patient being 38 years-old whereas theoldest patient was 88 years-old. The average age was lower in the PUpatient (60.18 years) and DFU patient (69.27 years) groups compared tothe patients in the VLU group (70.80 years). Regarding the gender of thethirty-three patients, as shown in Table 5 below, two-thirds of thepatients were males and one-third were females.

TABLE 5 Patients gender - all wounds Number of Gender patients % Male 6767.7 Female 32 32.3 Total 99 100

As shown in Table 6 below, each chronic wound type had representation inboth male and female populations for the ninety-nine patients presentedin the final analysis.

TABLE 6 Patients gender - number of patients by type of wound Gender PUDFU VLU Male 13 25 29 Female 2 7 23 Total 15 32 52

Other demographic information for the ninety-nine patients includedethnicity (all ninety-nine were Caucasian), smoking status as of thedate of screening (fifty-four never smoked, thirty-eight had stoppedsmoking, and seven patients were smoker), vital signs including heartrate, blood pressure, body temperature and respiration rate (none of theninety-nine patients vital signs was considered as clinicallysignificant by the investigators) and body-mass index

Among the three different types of chronic ulcers treated in the 99patients included in the final analysis, staging was assessed by theinvestigators at the patient's screening visit for PUs and DFUs as shownbelow in Tables 7 and 8, respectively.

The classification used for pressure ulcers was the staging systemproposed by the National Pressure Ulcer Advisory Panel (NPUAP) and theEuropean Pressure Ulcer Advisory Panel (EPUAP). (Source: NationalPressure Ulcer Advisory Panel and European Pressure Ulcer AdvisoryPanel. Pressure Ulcer Treatment. Quick Reference Guide 2009. Availableat: http://www.npuap.org). Only Stages II and III Pressure ulcers wereaccepted in the study. As shown on Table 8 (below), the majority (60%)of the PUs patients enrolled were classified as Stage III at studyentry.

TABLE 7 EPUAP/NPUAP Staging - Pressure Ulcers Stage DescriptionAdditional Description STAGE I Intact skin with nonblanchable The areamay be painful, firm, redness of a localized area soft, warmer or cooleras usually over a bony prominence, compared to adjacent tissue. Darklypigmented skin may not Stage I may be difficult to have visibleblanching; its color detect in individuals with dark may differ from thesurrounding skin tones. May indicate “at area. risk” persons (aheralding sign of risk). STAGE II Partial thickness loss of dermisPresents as a shiny or dry presenting as a shallow open shallow ulcerwithout slough or ulcer with a red pink wound bed, bruising*. This stageshould not without slough. May also present be used to describe skintears, as an intact or open/ruptured tape burns, perineal dermatitis,serum-filled blister. maceration or excoriation. *Bruising indicatessuspected deep tissue injury. STAGE III Full thickness tissue loss. Thedepth of a Stage III Subcutaneous fat may be visible pressure ulcervaries by but bone, tendon or muscle are anatomical location. The bridgenot exposed. Slough may be of the nose, ear, occiput and present butdoes not obscure the malleolus do not have depth of tissue loss. Mayinclude subcutaneous tissue and Stage undermining and tunneling. IIIulcers can be shallow. In contrast, areas of significant adiposity candevelop extremely deep Stage III pressure ulcers. Bone/tendon is notvisible or directly palpable. STAGE IV Full thickness tissue loss withThe depth of a Stage IV exposed bone, tendon or muscle, pressure ulcervaries by Slough or eschar may be present anatomical location. Thebridge on some parts of the wound bed. of the nose, ear, occiput andOften include undermining and malleolus do not have tunneling.subcutaneous tissue and these ulcers can be shallow. Stage IV ulcers canextend into muscle and/or supporting structures (e.g., fascia, tendon orjoint capsule) making osteomyelitis possible. Exposed bone/tendon isvisible or directly palpable. UNSTAGEABLE Full thickness tissue loss inUntil enough slough and/or which the base of the ulcer is eschar isremoved to expose the covered by slough (yellow, tan, base of the wound,the true gray, green or brown) and/or depth, and therefore stage, eschar(tan, brown or black) in cannot be determined. Stable the wound bed.(dry, adherent, intact without erythema or fluctuance) eschar on theheels serves as “the body's natural (biological) cover” and should notbe removed.

The classification used for DFUs was the University of Texasclassification. The University of Texas system assesses ulcer depth, thepresence of wound infection, and the presence of clinical signs oflower-extremity ischemia. This system uses a matrix of grade on thehorizontal axis and stage on the vertical axis. Only Diabetic footulcers with Stages 1A (superficial, non-infected, non-ischemic wound notinvolving tendon, capsules, or bone) or 2A (non-infected, non-ischemicwound penetrating to tendon or capsule but not in the bone or joint)were accepted. As shown on Table 11 (below), the majority (56.3%) of theDFUs enrolled were of Stage 2A.

TABLE 8 University of Texas Staging- Diabetic Foot Ulcers 0 1 2 3 A Pre-or post- Superficial Wound Wound ulcerative site wound not penetratingto penetrating that has healed involving tendon or bone or tendon,capsule, capsule joint or bone B With infection With infection Withinfection With infection C With ischemia With ischemia With ischemiaWith ischemia D With infection With infection With infection With andischemia and ischemia and ischemia infection and ischemia

No classification was used for VLUs due to the lack of standardizedstaging internationally recognized.

For the ninety-nine patients presented in the study, Table 9 presents abreakdown with respect to chronic wound type of the patients; themajority of the patients were of either the DFU or VLU groups.

TABLE 9 Type of wounds included in the final analysis Number ofPercentage Wound Type patients of patients DFU 32 33.3% PU 15 15.2% VLU52 52.5% Total Number of Patients 99  100%

The staging of the wounds for the PU and DFU patients presented in thefinal analysis is provided in Table 10 and Table 11, respectively.

TABLE 10 Pressure Ulcers' Staging at Screening Number of Stage patients% II 6 40.0 III 9 60.0 Total 15 100

TABLE 11 Diabetic Foot Ulcers' Staging at Screening Number of Stagepatients % 1A 14 43.8 2A 18 256.3 Total 32 100

Given that the study was an observational study in real-life conditions,age of the wounds was not part of the eligibility criteria therebyexplaining degree of variation from one wound to another when comparingpatient-to-patient for the chronic wound type.

On average, the duration of the chronic ulcers (PUs, DFUs and VLUscombined) at screening for the study was 35.5 months. Average chroniculcers duration was 64.1 months for PUs, 424.9 months for VLUs and 10.2months for DFUs. The youngest ulcer had happened just prior to thepatient's enrollment (PU) whereas the oldest ulcer was approximately 52years old (VLU). Table 12 below presents a summary (all woundscombined), while Table 13 presents a summary specifically for the (PUpatients, Table 14 presents a summary for the DFU patients and Table 15presents a summary for the VLU patients.

TABLE 12 Chronic ulcers duration at baseline (including PUs, DFUs, andVLUs) Average S.D. Minimum Maximum (months) (months) (months) (months)35.5 92.8 0.0 625.3 Note: N = 99. Duration calculated from date of wounddiagnosis to date of screening, as reported by patients. S.D.: StandardDeviation

TABLE 13 Pressure ulcers duration at screening Average S.D. MinimumMaximum (months) (months) (months) (months) 42.1 158.5 0.0 625.3 Note: N= 15. Duration calculated from date of wound diagnosis to date ofscreening, as reported by patients. S.D.: Standard Deviation.

TABLE 14 Diabetic foot ulcers duration at screening Average S.D. MinimumMaximum (months) (months) (months) (months) 10.2 14.7 0.0 72.5 Note: N =32. Duration calculated from date of wound diagnosis to date ofscreening, as reported by patients. S.D.: Standard Deviation.

TABLE 15 Venous leg ulcers duration at screening Average S.D. MinimumMaximum (months) (months) (months) (months) 424.9 93.9 0.0 518.0 Note: N= 52. Source: Duration calculated from date of wound diagnosis to dateof screening, as reported by patients. S.D.: Standard Deviation.

Further characteristics of the chronic wound type that each of theninety-nine patients presented in the final analysis were suffering fromincluded the area of skin and soft tissues that the given patient'swound encompassed and the bodily location of the wound. The size of thechronic ulcers was on average 10.96 cm² at the Screening visit, varyingfrom 0.1 cm² up to 52.2 cm². The median size (all wounds combined) atstudy entry was 8.85 cm² (see Table 16).

TABLE 16 Chronic ulcers (all types) areas at screening and FirstTreatment Visit Average S.D. Minimum Maximum (cm²) (cm²) (cm²) (cm²)7.39 9.47 0.1 52.50 Note: N = 99. S.D.: Standard Deviation.

The size of the chronic wound varied between the three chronic woundtypes, with DFUs presenting the smallest average size and VLUspresenting the largest average size at both the Screening and the FirstTreatment Visit time points. Wound size data for patients with PU, DFU,or VLU were presented in Tables 17, 18, and 19, respectively.

TABLE 17 Pressure ulcers areas at Screening and First Treatment VisitAverage S.D. Minimum Maximum (cm²) (cm²) (cm²) (cm²) 4.29 5.36 0.1 21.30Note: N = 15. S.D.: Standard Deviation.

TABLE 18 Diabetic foot ulcers areas at Screening and First TreatmentVisit Average S.D. Minimum Maximum (cm²) (cm²) (cm²) (cm²) 3.03 3.4 0.112.30 Note: N = 32. S.D.: Standard Deviation.

TABLE 19 Venous leg ulcers areas at Screening and First Treatment VisitAverage S.D. Minimum Maximum (cm²) (cm²) (cm²) (cm²) 10.96 11.39 0.352.5 Note: N = 52. S.D.: Standard Deviation

The bodily location of the chronic wounds in each of the three patientgroups, at the final analysis (ninety-nine patients) varied betweenpatients in the given group. The wound locations for the PU patients,the DFU patients and the VLU patients in the final analysis arepresented in Tables 20, 21 and 22, respectively.

TABLE 20 Pressure ulcers location Pressure Ulcers Total Number WoundLocation of Wounds Thigh 1 Sacrum/buttock 9 Heel 5 Total 15 Note: N =15.

TABLE 21 Diabetic foot ulcers location Total Number Diabetic foot ulcersof Wound Location Wounds 1st Dorsal Toe 2 1st Plantar Toe 4 4^(th)Plantar Toe 1 Ankle 5 Arch of Foot 2 Ball of Foot 13 Dorsal Foot 1 Heel4 Total 32 Note: N = 32.

TABLE 22 Venous leg ulcers location Total Venous leg ulcers Number ofWound Location Wounds External left ankle 6 External left leg 2 Externalright ankle 7 External right calf 4 External right leg 3 Internal leftankle 12 Internal left leg 1 Internal right ankle 9 Internal right calf3 Internal right leg 3 External left calf 1 Internal left calf 1 Total52 Note: N = 52.

All of the chronic ulcers treated in the study had failed on at leastone form of treatment before. Overall, the vast majority had beenpreviously treated with dressings and debridement, as per standard ofcare and clinical practice guidelines, with a complete list of priortreatments being presented in Tables 23 (all wounds), Table 24 (for thePU patients), Table 25 (for the DFU patients), and Table 26 (for the VLUpatients). Dressings and medicated dressings were the most frequenttreatments mentioned. Thirteen DFU patients had also already receivedsystemic antibiotics, and fourteen VLU patients had a history of failedskin graft.

TABLE 23 Ulcers' prior treatments (all wounds combined) Number ofDescription patients Topical antibiotics 7 Compression (bands, socks) 3Dressings (dry, wet, gels) 26 Medicated dressings 102 Systemicantibiotics 6 Topical disinfectants 9 (including topical ointments)Debridement 14 Grafting 44 Offloading 1 Collagen 13 Negative PressureWound 3 Therapy Note: N = 99. Patients might have more than onetreatment.

TABLE 24 Pressure ulcers' prior treatments Number of Descriptionpatients Topical antibiotics 2 Dressings (dry, wet, gels) 0 Medicateddressings 7 Systemic antibiotics 1 Topical disinfectants 2 TopicalOintments 3 Debridement 1 Grafting 0 Note: N = 15. Patients might havemore than one treatment.

TABLE 25 Diabetic foot ulcers' prior treatments Number of Descriptionpatients Topical antibiotics 4 Compression (bands, socks) 0 Dressings(dry, wet, gels) 5 Medicated dressings 20 Systemic antibiotics 4 Topicaldisinfectants 4 Grafting 1 Offloading 1 Note: N = 32. Subjects mighthave more than one treatment.

TABLE 26 Venous leg ulcers' prior treatments Number of Descriptionpatients Topical antibiotics 3 Compression (bands, socks) 3 Dressings(dry, wet, gels) 21 Medicated dressings 75 Systemic antibiotics 1Topical disinfectants 1 Topical Ointments 2 Debridement 13 Grafting 13Collagen 13 Negative Pressure Wound 3 Therapy Note: N = 52.

Patient compliance to study visits from Screening to the end of study,was to be considered as excellent, which is not always evident ascompliance may sometimes be an issue with patients affected by chronicwounds. Overall, 95.2% of study treatment visits planned by the protocolwere received during the treatment period.

With respect to the length of time that the patients were underinvestigational treatment, on average, patients were underinvestigational treatment for 80.45 days. The shortest period treatmentwas days, for one of the DFU patients, whereas the longest treatmentperiod was 224 days for four of the VLU patients. Table 27 presents theoverall data for the ninety-nine patients' duration of investigationaltreatment.

TABLE 27 Investigational treatment duration Average Standard MinimumMaximum treatment deviation duration duration duration (days) (days)(days) (days) 80.45 51.18 0.0 224.0 Note: N = 99.

The duration of investigational treatment varied depending on thechronic wound type group, and as shown in Table 28, the number oftreatment days in the VLU group was lower than in the groups of PU andVLU. It might be explained by the high rate of wound closure in the VLUgroup, and by the fact that these wounds responded overall quickly andfavourably to the study treatment.

TABLE 28 Investigational treatment duration, by type of wound AverageStandard Minimum Maximum treatment deviation duration duration Type ofwound duration (days) (days) (days) (days) PU 87.53 37.14 19 142 DFU70.28 66.6 0 224 VLU 84.67 43.13 3 173 Note: N = 99. Average of allwounds shown in Table 27.

A majority of the thirty-three patients comprising the interim analysisof the study responded positively to the treatment with the biophotoniccomposition; a total of twenty-one of the patients were considered to befull responders, wherein a full responder was defined as having adecrease of the wound size area of more than 90% at the end of the studyperiod and/or decrease of more than 50% of the size in 15 days or less.This cohort of full responders comprised twelve DFU patients, eight VLUpatients and one PU patient.

Furthermore, three patients in each of DFU and VLU wound types wereconsidered to be partial responders, wherein a partial responder wasdefined s having a decrease of the size of the wound during the studyperiod, but without meeting the criteria of full responder. Nine of thefull responder DFU patients experienced a total closure of their woundsby the end of their participation in the study, while seven of the eightVLU experienced full closure by the end of their participation (with theeighth full responder VLU experiencing a 97% decrease in wound area bythis patients end of participation).

The full responder PU patient, with this patient experienced a completewound closure by the day of their last treatment visit (day 47).Overall, of the thirty-three patients presented in the interim analysis,16 patients (48.5% of the total number of patients) experienced a fullclosure of their wound totally during the study period, and the meantime to reach total closure was 46.8 days. This mean time varieddepending on the type of wound; it was lower for DFUs (mean time of 37.2days), whereas it was higher for PUs (47.0 days) and VLUs (53.6 days).Of the three partial responders in the VLU cohort, two of thesepatients' wounds were graft-ready by their completion of the study, andfor the three partial responders in the DFU patient cohort, one of thesepatients' wound was graft ready by the end of the patient'sparticipation in the study.

In the ninety-nine patient study, the average number of treatments bytypes of wounds are (i) 23.90 treatments for patients with VLU, (ii)18.31 treatments for patients with DFU, and (iii) 23.40 treatments forpatients with PU. Significant variations of the wound size area ascompared to baseline were found for VLU (p<0.001) and DFU (p=0.001). Inthe study, 47 wounds closed completely during the study period,especially VLUs (26) and DFUs (17). It represents 47.5% of the woundstreated during the study period. By type of wounds, the results are (i)50% of the VLU wounds closed completely; (ii) 50% of the DFU woundsclosed completely; and (iii) 33.3% of the PU wounds closed completely.

Looking at the trajectories of relative wound area regression over time,superior results were obtained with VLUs and DFUs, showing similarresults. The mean time to reach a regression of 50% of the wound areawas approximately 8 weeks for VLUs and 3.5 weeks for DFUs.

Wound breakdown (dehiscence) was assessed at least two weeks afterconfirmation of wound closure through the observations performed duringthe follow-up period. This phase of the study was specifically designedto allow the documentation of the wound closure persistence based on thedefinition of wound closure being “wound closure is defined as skinre-epithelialization without drainage or dressing requirements confirmedat two consecutive visits, two weeks apart”). Incidence of woundbreakdown was assessed at two timepoints during the follow-up period:

-   -   after the two-week evaluation period following wound closure:        two wounds (5.13%) had a wound breakdown (N=39);    -   after the eight-week evaluation period following wound closure:        two wounds (5.71%) had a wound breakdown (N=35).

These low percentages of wound breakdown show that, when a chronic woundtotally closed following treatment, 95% of them remain totally closedafter the two-week follow-up period, and even after the eight-weekfollow-up period.

This low incidence of wound breakdown at two weeks and eight weeks'post-closure confirms the efficacy profile and long-lasting action of abiophotonic composition of the present disclosure on wound closure.

From the 47 wounds that closed completely, four wounds closed at thelast study visit and it was therefore not possible to confirm theabsence of wound breakdown for these wounds. Thirty-four wounds werereassessed at Follow-up visit 1 (two weeks following wound closure) and35 at Follow-up visit 3 (8 weeks following wound closure). During thesevisits, only two wounds reopened, meaning a rate of wound breakdownafter 8 weeks of 5%.

Table 29 presents the results of the wound breakdown.

TABLE 29 Wound breakdown investigation for closed wounds Wound breakdownWound breakdown Wound at two weeks at eight weeks type Closed wounds (N= 34) (N = 35) VLU 26 0 1 (0603) DFU 17 0 1 (1310) PU 4 0 0 All wounds47 0 2 Note: N = 47.

Even if a wound was not closed, it was of interest to know if the woundbed was prepared enough to receive a skin graft. For this purpose, aspecific question was added in the study questionnaire for ulcerassessment. The results in terms of wound bed preparation were alsopositive in this study. Investigators were asked to assess regularlyduring the study period if the wounds became ready to skin graft. Evenif the wounds had a profile of hard to heal non responsive wounds atstudy entry, with a high rate of prognosis factors of poor healing, morethan two thirds of the VLU (69.2%) and DFU (68.8%) wounds became readyto skin graft at one point during the study period. Table 30 presentsthese different data by type of wound, and overall. An estimate of themean and median age to become graft ready was estimated for each type ofwound using the Kaplan-Meir method. Overall, mean time was 95.7 days andmedian time 86.0 days, but with a significant difference between thewounds. The fastest were the DFUs (mean time of 79.0 days and mediantime of 41.0 days), followed by VLUs and then by PUs.

TABLE 30 Number of wounds becoming “Graft ready”, after initiation ofthe treatment with the biophotonic composition - summary by type ofwound Ready for graft (invest opinion) Yes No Total Wound type N % N % N% VLU 36 69.2% 16 30.8% 52 100.0% DFU 22 68.8% 10 31.3% 32 100.0% PU 640.0% 9 60.0% 15 100.0% All wounds 64 64.6% 35 35.4% 99 100.0% Note: N =99.

Local clinical signs of wound colonization over time:

Investigators were asked at the first Treatment visit to assessdifferent aspects of the wound and peri-wound skin, including thepresence of local signs of colonization or infection (Redness, Pain,Heat, Pus and Swelling). This assessment, which is generally consideredas a sign of inflammation, was carried out at the first treatment visitand at every study treatment.

General characteristics at first treatment visit were:

-   -   As expected with these hard to heal chronic wounds, the majority        of them had local clinical signs of bacterial colonization at        study entry, with redness (37.4% all wounds combined) and heat        (7.1%). It concerned especially VLU, with 57.7% having redness        and 11.5% having heat. There was no clinical sign of bacterial        colonization for DFUs and PUs;    -   This hypothesis of a probable colonization of some of the wounds        at study entry is reinforced by the amount of exudate: 26.9% of        high exudate level for VLUs and 20.0% for DFUs (18.2% overall);    -   The great majority (98.0%) had no visible presence of pus.

A fast regression of these clinical signs of bacterial colonization wasobserved as soon as Week 4:

-   -   Redness: from 58% to 36%;    -   Pain: from 52% to 16%;    -   Swelling: from 29% to 14%;    -   No more wound with pus.

Complete results of these different assessments at the first treatmentvisit are presented in the table below.

TABLE 31 Local signs of colonization/Infection by wound type at 1^(st)Treatment Visit Wound type VLU DFU PU Total N % N % N % N % Redness YES30 57.7% 6 18.8% 1 6.7% 37 37.4% NO 22 42.3% 26 81.3% 14 93.3% 62 62.6%Total 52 100.0% 32 100.0% 15 100.0% 99 100.0% Pain YES 27 51.9% 1 3.1% 00.0% 28 28.3% NO 25 48.1% 31 96.9% 15 100.0% 71 71.7% Total 52 100.0% 32100.0% 15 100.0% 99 100.0% Heat YES 6 11.5% 1 3.1% 0 0.0% 7 7.1% NO 4688.5% 31 96.9% 15 100.0% 92 92.9% Total 52 100.0% 32 100.0% 15 100.0% 99100.0% Pus YES 1 1.9% 1 3.1% 0 0.0% 2 2.0% NO 51 98.1% 31 96.9% 15100.0% 97 98.0% Total 52 100.0% 32 100.0% 15 100.0% 99 100.0% Swell- YES15 28.8% 3 9.4% 3 20.0% 21 21.2% ing NO 37 71.2% 29 90.6% 12 80.0% 7878.8% Total 52 100.0% 32 100.0% 15 100.0% 99 100.0% Note: N = 99.

As was determined previously for the interim analysis, no case of woundinfection was observed in DFU patients among the 17 subjects included inthe interim analysis. Additionally, no DFU wound required a local orsystemic antimicrobial therapy during the interim study period. Asmentioned in literature (Lavery et al. (2006), “The efficacy and safetyof Grafix(®) for the treatment of chronic diabetic foot ulcers: resultsof a multi-centre, controlled, randomised, blinded, clinical trial.”International Wound Journal, 11(5):554-60), foot infections occurfrequently in individuals with diabetes, with the well-known risk oflower-extremity amputations (infected foot wounds precede two-thirds oflower-extremity amputations, according to Lavery et al., 2006). Woundinfections are also responsible of delayed wound healing and woundbreakdowns.

This very low number of wound infections throughout the final studyperiod is in favour of an action of the treatment on control of thewound biofilm.

While preferred embodiments of the disclosure have been described aboveand illustrated in the accompanying drawings, it will be evident tothose skilled in the art that modifications may be made therein withoutdeparting from the essence of this disclosure. Such modifications areconsidered as possible variants comprised in the scope of the subjectmatter of this disclosure.

INCORPORATION BY REFERENCE

All references cited in this specification, and their references, areincorporated by reference herein in their entirety where appropriate forteachings of additional or alternative details, features, and/ortechnical background.

EQUIVALENTS

While the disclosure has been particularly shown and described withreference to particular embodiments, it will be appreciated thatvariations of the above-disclosed and other features and functions, oralternatives thereof, may be desirably combined into many otherdifferent systems or applications. Also, that various presentlyunforeseen or unanticipated alternatives, modifications, variations orimprovements therein may be subsequently made by those skilled in theart which are also intended to be encompassed by the followingembodiments.

1. A method for inhibiting biofilm formation, or disrupting existing ordeveloping biofilms in a subject, comprising: a) topically applying acomposition comprising at least one chromophore and a pharmaceuticallyacceptable carrier; and b) illuminating said composition with actiniclight, thereby inhibiting biofilm formation, or disrupting existing ordeveloping biofilms in the subject.
 2. The method according to claim 1,wherein the composition is a biophotonic composition.
 3. The methodaccording to claim 1, wherein the at least one chromophore is selectedfrom the group consisting of a xanthene derivative dye, an azo dye, abiological stain, and a carotenoid.
 4. The method according to claim 1,wherein the at least one chromophore is selected from the groupconsisting of eosin, erythrosine, and Saffron red powder.
 5. The methodaccording to claim 1, wherein at least one chromophore is eosin Y oreosin B.
 6. The method according to claim 1, wherein at least onechromophore is eosin Y.
 7. The method according to claim 1, wherein atleast one chromophore is erythrosine B. 8.-9. (canceled)
 10. The methodaccording to claim 1, wherein the at least one chromophore is present inan amount of from about 0.02% to about 8% by weight of the composition.11.-13. (canceled)
 14. The method according to claim 1, wherein the atleast one chromophore is present in an amount of from about 0.5% byweight of the composition. 15.-33. (canceled)
 34. The method accordingto claim 1, wherein the composition further comprises at least onehealing factor.
 35. The method according to claim 34, wherein thehealing factor is selected from the group consisting of hyaluronic acid,glucosamine, and allantoin. 36.-55. (canceled)
 56. The method accordingto claim 1, wherein said composition is illuminated with actinic lightfor at least one treatment period of from about 1 minute to about 9minutes per cm² of an area to be treated.
 57. The method according toclaim 1, wherein said composition is illuminated with actinic light forat least one treatment period of from about 2 minutes to about 8 minutesper cm² of an area to be treated. 58.-60. (canceled)
 61. The methodaccording to claim 1, wherein said composition is illuminated withactinic light for at least two treatment periods, each period followedby a resting interval.
 62. (canceled)
 63. The method according to claim1, wherein said composition is illuminated with at least two treatmentperiods of actinic light wherein each treatment period is from about 1minute to about 5 minutes per cm of an area to be treated, wherein eachtreatment period is followed by a resting interval for about 1 minute toabout 5 minutes.
 64. The method according to claim 1, furthercomprising: a) topically applying the composition to the subject's areaof biofilm; b) illuminating the subject's area of biofilm with actiniclight for a period of from about 1 minute to about 10 minutes; c)removing the source of actinic light away from the subject's area ofbiofilm treated for a period of from about 1 minute to about 5 minutes;d) illuminating the subject's area of biofilm with actinic light for asecond time period of from about 1 minute to about 10 minutes; andwherein the first illumination of actinic light activates thecomposition. 65.-67. (canceled)
 68. The method according to claim 1,wherein the biofilm is associated with at least one of the subject'sskin and the subject's soft tissues, and comprises at least one ofStaphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis.69.-73. (canceled)
 74. The method according to claim 1, wherein thebiofilm comprises one of gram-negative bacteria and gram-positivebacteria.
 75. The method according to claim 1, wherein the biofilmcomprises gram-positive bacteria. 76.-92. (canceled)